Sequencing by synthesis using mechanical compression

ABSTRACT

Methods and apparatuses for sequencing by synthesis using mechanical compression. These methods and apparatuses may mechanically control microfluidic movement using a force applicator and an elastically deformable sheet.

CLAIM OF PRIORITY

This patent application claims priority to U.S. Provisional Pat.Application No. 63/298,973, titled “MICROFLUIDIC TWO-DIMENSIONALCAPILLARY MANIPULATION DEVICES AND METHODS,” filed on Jan. 12, 2022,U.S. Provisional Pat. Application No. 63/393,815, titled “MECHANICALMICROFLUIDIC MANIPULATION DEVICES AND METHODS,” filed on Jul. 29, 2022,U.S. Provisional Pat. Application No. 63/417,302, titled “MECHANICALMICROFLUIDIC MANIPULATION DEVICES AND METHODS,” filed on Oct. 18, 2022,and U.S. Provisional Pat. Application No. 63/418,028, titled “MECHANICALMICROFLUIDIC MANIPULATION DEVICES AND METHODS,” filed on Oct. 20, 2022.Each of these applications is herein incorporated by reference in itsentirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

BACKGROUND

Microfluidics deal with very small volumes of fluids, down tofemtoliters (fL) which is a quadrillionth of a liter. Fluids behave verydifferently on the micrometric scale than they do in everyday life:these unique features are the key for new scientific experiments andinnovations. Microfluidic devices may include micro-channels and requiremicrominiaturized devices containing chambers and tunnels through whichfluids flow or are confined.

For example, digital microfluidics (DMF) is a powerful technique forsimple and precise manipulation of microscale droplets of fluid. DMF hasrapidly become popular for chemical, biological, and medicalapplications, as it allows straightforward control over multiplereagents (no pumps, valves, or tubing required), facile handling of bothsolids and liquids (no channels to clog), and compatibility with eventroublesome reagents (e.g., organic solvents, corrosive chemicals)because hydrophobic surfaces (typically Teflon-coated) in contact withthe droplets of fluid are chemically inert. However, conventional DMFdevices use relatively large electric fields selectively applied to anarray of electrodes to manipulate the droplets. The generation andcontrol of these electric fields requires specialized and complexcircuitry capable of withstanding the relative high voltages.

SUMMARY OF THE DISCLOSURE

Described herein are methods of performing sequencing by synthesis usingmechanical compression to manipulate droplets within an air gap formedbetween a first elastically deformable sheet and a second sheet. Thefirst sheet and the second sheet may each include hydrophobic andoleophobic surfaces facing the air gap. Either or both surfaces may befunctionalized, e.g., to include hybridization regions, such as primerhybridization region). In general, these methods may include the use ofa mechanical force actuator that can be driven to locally deform thefirst, elastically deformable sheet to form localized region of lowergap width adjacent to a droplet and can be moved along the surface ofthe first sheet to pull the droplet after it to translate the region oflocally reduced gap width. The droplet will follow the region of lowergap width within the air gap by capillary action. Also described hereinare apparatuses (e.g., devices and systems, including cartridges) forperforming any of these methods for sequencing by synthesis.

In general, these methods and apparatuses may be used for preparing,manipulating and/or analyzing fluidic droplets, such as microfluidicdroplets. For example, described herein are microfluidic apparatusesthat may be especially helpful for handling and analyzing a clinical,laboratory, biological, or chemical samples. These apparatuses maygenerally operate by applying a mechanical force, such as a compressionforce, to an elastically deformable sheet that at least partially coversan air gap over a subregion of the air gap, to reduce the height of theair gap in a region that is adjacent to a droplet, causing the dropletto move towards this region of reduced height. By controlling therelative height of the air gap near the droplet (e.g., by controllingthe application of force to deform the elastically deformable sheet), adroplet may be efficiently and quickly moved around the air gap,allowing processing of the droplet including combining the droplet,dividing the droplet, mixing the droplet, cooling/heating the droplet(e.g., thermocycling the droplet), using magnetic particles within thedroplet (e.g., to bind/remove materials from the droplet) and the like.

Described herein are apparatuses (systems, devices, etc.) forcontrolling microfluidic movement, e.g., droplet movement) on a surfaceby mechanical means. These apparatuses may be referred to herein asmechanical microfluidics actuation devices (“mechanical microfluidicsactuators”) and may include a force applicator for applying force to anelastically deformable sheet that at least partially encloses an air gapwithin which one or more droplets resides. The elastically deformablesheet may be a part of the mechanical microfluidics actuator apparatus,or it may be part of a separate or integrated cartridge that is operatedon by the mechanical microfluidics actuator apparatus. In some examplesthe cartridge may include a first (e.g., upper) elastically deformablesheet, a second (e.g., lower) sheet which are held apart from each other(e.g. by a frame) to form an air gap within which one or more dropletsmay manipulated by the force applicator (e.g., a stylus, etc.). Themechanical microfluidics actuator may include force applicator, a forceapplicator driver subassembly (e.g., a force applicator subassembly),and thermal sub-assembly for controlling the temperature of one or moreregions of the air gap. Any of these mechanical microfluidics actuatorapparatuses may also include a magnetic control sub-assembly forcontrollably applying a magnetic field within the air gap. In someexamples, the apparatus may include a cartridge holder for securing thecartridge to a cartridge seat or seating region of the mechanicalmicrofluidics actuator. Optionally the apparatuses may include avacuum/suction sub-assembly for securing the cartridge to the seatingregion of the mechanical microfluidics actuator. In some examples themechanical microfluidics actuator apparatus may include a fluid handling(e.g., pipetting) sub-assembly for adding and/or removing fluid from theair gap. Other sub-assemblies forming a part of the mechanicalmicrofluidics actuator apparatus may include imaging sub-assemblies(e.g., for imaging droplets within the air gap) and/or for sensingsub-assemblies (e.g., for sensing droplets or other inputs from the airgap and mechanical microfluidics actuator). The mechanical microfluidicsactuator apparatuses described herein may also include one or morecontrol inputs (e.g., keyboards, touchscreens, buttons, switches, etc.)and/or one or more outputs (e.g., displays, LEDs, wirelesscommunications outputs/inputs, etc.) and hardware, software and/orfirmware for controlling these. In some cases the same features may beused for control inputs and outputs. In general, the mechanicalmicrofluidics actuators described herein may include one or morecontrollers for controlling and coordinating operation of the varioussub-assemblies.

For example, described herein are microfluidics apparatuses including: acartridge comprising a first sheet and a second sheet, wherein the firstand second sheets are secured opposite and approximately parallel at apredetermined distance relative to each other with an air gaptherebetween; a controller configured to selectively reduce thepredetermined distance in one or more regions within the air gapadjacent to a fluidic droplet positioned within the air gap to move thefluidic droplet within the air gap of the cartridge. The controllerand/or cartridge may be part of a mechanical microfluidics actuator; insome examples the cartridge may be separate from the mechanicalmicrofluidics actuator and may be removable from the mechanicalmicrofluidics actuator. Cartridges may be single-use or reusable (e.g.,washable, sterilizable, etc.). In some cases the cartridge may beintegrated into the mechanical microfluidics actuator.

The second sheet may be elastically deformable or non-deformable. Insome examples the second sheet is made of the same material as the firstsheet (e.g., an elastic material). The second sheet may be configured tobe secured (via suction) to the system holding the cartridge so that itmakes consistent thermal contact with the second sheet, so that thetemperature may be rapidly and efficiently changed by heating/cooling alocalized region (thermally controlled region) of the second sheet toheat/cool a droplet within the air gap, in a region overlying thethermally controlled region. In some examples the droplet may be movedand/or pinned by deforming the lower sheet to change the height of thelocal region of the air gap; alternatively or additionally, the dropletmay be moved and/or pinned by deforming the upper sheet to change theheight of the local region of the air gap. When the force deforming thesheet (either or both first and second sheets) is removed, the sheetsmay return to the neutral, undeformed state, so that the air gap returnsto approximately the same predetermined distance.

In any of these examples, the controller may be configured toselectively reduce the predetermined distance in the one or more regionswithin the air gap without reducing the predetermined distance in one ormore adjacent regions. The sheet may comprise a first surface facing theair gap. In general, either or both the surfaces of the air gap may behydrophobic and oleophobic; for examples, the first surface may behydrophobic and oleophobic, and/or the plate may include a surfacefacing the air gap that is hydrophobic and oleophobic. In general, thesurfaces (or materials forming the sheet(s)) described herein may beoleophobic in addition to hydrophobic.

In any of these examples, the air gap may include at least oneinput/output port. For example, the sheet and/or the plate may beconfigured to introduce a first fluidic droplet into the air gap byincluding one or more input and/or output ports. In some examples thefluidic droplet may be inserted into (or removed out of) the air gap.The inlet/outlet port may be a region of the first sheet that is cutaway. This cut-away (opening) may be between 1 mm and 10 cm long andbetween 1 mm and 10 cm wide (e.g., between 5 mm and 7 cm long andbetween 5 mm and 4 cm wide, etc.). The opening through the first sheetmay be offset from the edge of the sheet, e.g., by 5 mm or more (e.g.,by 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 1 cm or more,1.5 cm or more, 2 cm or more, etc.). The inlet/outlet port may beconfigured so that tension is maintained on the sheet in the regionsurrounding the inlet/outlet. Thus, the edge of the inlet/outlet may betensioned.

The air gap may generally be any appropriate height (“thickness”) whenforce is not being applied to the sheet. For example, the air gap mayhave a height of between about 0.4 mm and about 7 mm (e.g., betweenabout 0.5 mm and about 6 mm, between about 0.5 mm and about 5 mm,between about 0.5 mm and 4 mm, etc.). In general the apparatusesdescribed herein (including, but not limited to cartridges) may includean air gap formed between the first (e.g., upper) and second (e.g.,lower) sheets. The controller may be configured to apply a compressionforce to the sheet to selectively reduce the predetermined distance.

For example, described herein are microfluidics apparatuses comprising:a cartridge comprising a first sheet and a second sheet, wherein thefirst sheet and the second sheet are secured opposite and approximatelyparallel at a predetermined distance relative to each other with an airgap therebetween; a controller configured to selectively reduce thepredetermined distance in one or more regions within the air gapadjacent to a fluidic droplet positioned within the air gap to move thefluidic droplet within the air gap of the cartridge.

The controller may generally be configured to selectively reduce thepredetermined distance in the one or more regions within the air gapwithout reducing the predetermined distance in one or more adjacentregions by applying force (pressure) to elastically deform the firstsheet (e.g., the upper sheet). The first sheet may comprise a firstsurface facing the air gap, wherein the first surface is hydrophobic;the second sheet may comprise a second surface facing the air gap, andthe second surface facing the air gap may also be hydrophobic andoleophobic. The cartridge further may include at least one input port onthe first sheet and/or the second configured to introduce a firstfluidic droplet into the air gap.

The controller may generally be configured to apply (and move) acompression force across the sheet to selectively reduce thepredetermined distance so that the width of the air gap changesdynamically in a manner that draws a droplet to follow the reducedheight of the air gap as it moves across the sheet. The movement may becontinuous or periodic.

In general, the first (and/or second) sheet is configured to elasticallydeform in response to compression forces. In any of these methods andapparatuses, the first sheet and/or the second sheet may be partially orcompletely clear (e.g., optically transparent) or opaque depending. Insome cases both the first and second sheets are clear. In some examplesjust the second sheet is clear. In some examples, just the first sheetis clear.

As used here the sheet includes at least one surface that extends in aplane (e.g., in an x, y direction). In some examples the sheet may berelatively thin, as in the elastically deformable first sheet. In someexamples the sheet may include regions of different thicknesses. Thesecond sheet may not be elastically deformable. In some cases the secondsheet is rigid. In some cases the second sheet may have regions ofdifferent thicknesses. In some examples, the second sheet may beelastically deformable. In some examples the second sheet may bereferred to as a layer, plate, base, or the like.

Any of these apparatuses may include a force applicator (e.g., in someexamples a stylus) positioned to apply a force to an outer surface ofthe first sheet to reduce the height of the air gap in a local region,wherein the controller may be configured to command movement of theforce applicator relative to the outer surface (e.g., so that it movesacross the outer surface). The force applicator may be a mechanicalforce applicator (e.g., stylus, roller, ball point, etc.) and/or apressure applicator (e.g., jet applicator, etc.). The force applicatormay be contact or non-contract. In some examples the force applicatormay comprise a source of pressurized fluid.

As mentioned, as least one of the first or second sheets may beelastically deformable. In some examples the first sheet may be referredto as the top or upper sheet and the second sheet may be referred to asa bottom or lower sheet. The second sheet may also be referred to as aplate in some examples.

In apparatuses including a cartridge, the cartridge may include a frameto which the first sheet (and/or the second sheet) is tensioned tomaintain the first sheet opposite and approximately parallel to thesecond sheet. The frame may include a tensioner. In some examples thetension may be applied at the time of fabrication and the sheet may beaffixed (e.g., mechanically and/or chemically, e.g., by an adhesive) tothe frame under tension. The frame may surround the periphery of thecartridge. In some examples the cartridge may be divided up intoregions, including lanes, chambers, etc. that may be separated by one ormore walls (e.g., dividers, etc.). The dividers may be fixed (e.g.,adhesively attached, welded, etc.) to the upper and/or lower sheets. Insome examples the frame and the spacers are combined into a singlecomponent, referred to herein as a spacer frame.

The second sheet may be the same material as the first sheet (e.g., anelastic material) or it may be a different material. In some examplesthe second sheet is formed of an inelastic material. In some examplesthe second sheet is formed of a material is more rigid or stiff than thefirst sheet. The second sheet may be referred to in some examples as aplate. In any of the apparatuses and methods described herein the secondsheet maybe relatively thermally conductive to allow heatingtherethrough. In some examples the first sheet and the second sheet bothcomprise an elastic material held in tension to a frame of thecartridge. The first and second sheets may be any appropriate thickness,and may be the same thickness, or may be different thicknesses. Forexample, the first and/or second sheet may be between 0.05 mm and 5 mmthick (e.g., between about 0.075 mm and 2 mm, between about 0.1 mm and 5mm, between about 0.1 mm and 4 mm, between about 0.2 mm and 4 mm,between about 0.1 mm and 3.5 mm, between about 0.2 mm and 3.5 mm, etc.).

The first and second sheets may be formed of any appropriate material.In any of these apparatuses and methods the first and second sheet areformed of an elastomeric material. In some examples, the first sheet isa polyester material (e.g., TPE, TPU, etc.).

Any of these apparatuses may include a controller that is configured tooperate the force applicator to apply the force (e.g., a “compressionforce”) against the first or second sheets to reduce the height of aportion of the air gap adjacent to a droplet in order to cause thedroplet to move (e.g., by capillary action) to the region of reducedheight. The controller may generally control the force applicator,including controlling the z-height, e.g., based on the height of the airgap, and/or the x and/or y movement of the force applicator along theouter surface of the first (or in some examples, the second) sheet. Asthe force applicator is moved along the upper sheet, it may drivemovement of the droplet which may follow the lower-height region of theair gap formed by the moving force applicator.

The controller may receive input from one or more sensors (e.g., opticalsensor, electrical sensors, force sensors, pressure sensors, etc.)including sensors identifying the position of the droplet. In someexamples the force applicator includes one or more sensors. Thecontroller may be configured to execute pre-programmed and/or dynamicsteps, including moving the force applicator in a pattern in order toachieve one or more fluid handling maneuvers, such as splitting ordividing a droplet, combining a droplet, mixing a droplet, washing adroplet (or a material, such as a magnetic material, magnetic beads,etc. within the droplet), etc. For example the controller may beconfigured to apply a pinning compression force to divide the firstfluidic droplet and to apply an actuation compression force proximate tothe pinning compression force to elongate and form a second fluidicdroplet from the first fluidic droplet and wherein the pinningcompression force is greater than the actuation compression force. Thecontroller may be configured to apply a pinning compression force todivide the first fluidic droplet and to apply an actuation compressionforce proximate to the pinning compression force to elongate and form asecond fluidic droplet from the first fluidic droplet and wherein thepinning compression force is greater than the actuation compressionforce.

In some examples the controller is configured to: apply a compressionforce to the sheet between two or more separate fluidic droplets; andrelease the compression force to combine the two or more separatefluidic droplets into a single fluidic droplet. In some examples thecontroller is configured to alternately apply a first compression forceand a second compression force different than the first compressionforce to the sheet to mix together two or more separate fluidicdroplets. Thus, the controller may be configured to mix together two ormore fluidic droplets by repeatedly applying and releasing a compressionforce to the sheet adjacent to the two or more fluidic droplets.

The controller may also be configured to coordinate one or moreprocesses in addition to controlling movement of the force applicator.For example, the coordinator may control and/or coordinate theadditional of fluid into the air gap (e.g., fluid dispensing, which maybe manually, automatically or semi-automatically performed.). Acontroller may also control or coordinate the activity of one or moreheaters, magnets, etc. For example, in some examples the controller isconfigured to control a magnet to attract ferrous particles suspendedwithin the first fluidic droplet. The controller may be furtherconfigured to re-suspend one or more ferrous particles in the fluidicdroplet by applying and releasing a compression force to the fluidicdroplet and disabling a magnet. The first sheet may further comprise twoor more pinning posts disposed on the surface and extending into thegap, the pinning posts being configured to restrict movement of thefirst fluidic droplet. Any of these apparatuses may include a heaterdisposed under the second surface opposite the two or more pinningposts. Alternatively, the apparatuses described herein may be usedwithout posts.

Any of the apparatuses described herein may include a well into whichthe droplet may be moved. The well may be part of the cartridge and/orpart of the mechanical microfluidics actuation device (“mechanicalmicrofluidics actuator”). In some examples the well is formed on a baseor seating region of the mechanical microfluidics actuator. For example,the cartridge may seat in the seating region of the mechanicalmicrofluidics actuator and may conform to a shape of the seating regionincluding one or more wells. In some examples the well is part of thecartridge, for example, the well is configured to restrict movement ofthe fluidic droplet.

Any of these apparatuses (e.g., mechanical microfluidics actuatorapparatuses) may include base having a cartridge seat configured tosecure the cartridge so that the second sheet is held against cartridgeseat with at least a region of the cartridge in communication with aheating element disposed beneath the seat and configured to heat thefluidic droplet within the air gap. In some examples the mechanicalmicrofluidics actuator apparatus may include a securement, such as aclamp, for securing the cartridge to and/or in the seat. In someexamples the seat may include a plurality of suction ports to apply anegative pressure to hold the cartridge (e.g., the second or lower sheetof the cartridge) to the seat. The use of suction may be particularlybeneficial to secure the cartridge in place snugly so that good thermalcontact is maintained. This use of suction may also allow transfer ofthree-dimensional structures (e.g., wells, ridges, shelves, etc.) to thecartridge (e.g., the lower sheet of the cartridge may conform to theshape of the seat).

In examples having wells, the heaters, magnets, etc. may be configuredto effect the well. In some examples the heater and/or magnet may bearranged to effect a droplet within the air gap in a region that is notpart of a well, including in a rail region. In general, the heaterand/or magnet may be part of the cartridge or may be part of theactuation device (e.g., drive unit).

Also described herein are methods of manipulating one or moremicrofluidic droplets using mechanical actuation of droplets. Forexample, described herein are methods including: introducing a firstfluidic droplet into an air gap formed between: a first sheet having afirst surface that is hydrophobic and oleophobic and a second sheethaving a second surface that is hydrophobic and oleophobic, wherein thefirst sheet and the second sheet are secured opposite and approximatelyparallel at a predetermined distance relative to each other with an airgap therebetween; and applying a force (e.g., mechanical force) toelastically deform the first sheet to reduce the distance of the air gapbetween the first sheet and the second sheet in a region within the airgap that is adjacent to the fluidic droplet to move the fluidic dropletwithin the air gap. Applying force may comprise moving the force alongthe outer surface of the sheet to selectively reduce the distancebetween the first sheet and the second sheet so that the droplet followsthe applied force. In any of these examples applying the force maycomprise moving a stylus against the first sheet. Applying the force maycomprise driving movement of a pressure applicator to apply acompression force to an outer surface of the first sheet, wherein thepressure applicator is controlled by a controller. Any of these methodsmay include forming a second fluidic droplet from the fluidic dropletby: applying a pinning compression force to the first sheet to dividethe fluidic droplet; and applying an actuation compression force to thefirst sheet proximate to the pinning compression force to elongate andform the second fluidic droplet, wherein the pinning compression forceis greater than the actuation compression force.

For example, a method of microfluidically manipulating a droplet mayinclude: introducing the droplet into an air gap formed between a firstsheet that is elastically deformable and a second sheet, wherein thefirst sheet is spaced opposite from the second sheet to form an air gaphaving a gap width of a predetermined distance in a neutral state;applying a compression force against the first sheet using a mechanicalforce applicator to form a region of locally reduced gap width withinthe air gap that is adjacent to droplet, thereby drawing the droplettowards the region of locally reduced air gap; and moving the dropletwithin the air gap by translating the mechanical force applicator alongan outer surface of the first sheet to translate the region of locallyreduced gap width within the air gap so that the droplet follows themechanical force applicator.

In any of these methods moving the droplet may comprise moving thedroplet along a rail region of the air gap, wherein the rail region hasa gap width that is less than the gap width of a region of the air gapsurrounding the rail region.

For example a method of microfluidically manipulating a droplet mayinclude: introducing the droplet into an air gap formed between a firstsheet that is elastically deformable and a second sheet, wherein thefirst sheet is spaced opposite from the second sheet to form an air gaphaving a gap width of a predetermined distance in a neutral state,wherein the air gap is open to atmospheric pressure and unpressurized,further wherein the droplet positioned in a rail region of the air gaphaving a gap width that is less than the gap width of a regionsurrounding the rail region; applying a compression force against thefirst sheet using a mechanical force applicator to form a region oflocally reduced gap width within the air gap that is adjacent todroplet, thereby drawing the droplet towards the region of locallyreduced air gap by capillary action; and moving the droplet along therail region of the air gap by translating the mechanical forceapplicator along an outer surface of the first sheet to translate theregion of locally reduced gap width within the air gap and thereby pullthe droplet within the air gap.

Any of these methods may include moving the droplet into a well formedby the second sheet. The well may be used to modify the droplet. Forexample, the well may be used to heat/cool the droplet, and/or to reactmaterial within the droplet. The methods described herein may includecontrolling a temperature of the well. For example, a method ofmicrofluidically manipulating a droplet may include: introducing thedroplet into an air gap formed between a first sheet that is hydrophobicand oleophobic and is that is elastically deformable, and a second sheetthat is hydrophobic and oleophobic, wherein the first sheet is spacedopposite from the second sheet to form an air gap having a gap width ofa predetermined distance in a neutral state, wherein the air gap is opento atmospheric pressure and unpressurized; applying a compression forceagainst the first sheet using a mechanical force applicator to form aregion of locally reduced gap width within the air gap that is adjacentto droplet, thereby drawing the droplet towards the region of locallyreduced air gap by capillary action; and moving the droplet into a wellformed by the second sheet by translating the mechanical forceapplicator along an outer surface of the first sheet to translate theregion of locally reduced gap width within the air gap and thereby pullthe droplet within the air gap and into the well; modifying the dropletwithin the well; and moving the droplet out of the well by translatingthe mechanical force applicator along the outer surface of the firstsheet to translate the region of locally reduced gap width within theair gap away from the well and thereby pull the droplet out of the well.Any of these methods may include moving the droplet out of the well bytranslating the mechanical force applicator along the outer surface ofthe first sheet to translate the region of locally reduced gap widthwithin the air gap away from the well and thereby pull the droplet outof the well.

Thus, in general, any of these methods may include modifying the dropletwithin the air gap. Modifying may include one or more of: reacting oneor more materials within the droplet, heating the droplet, addingmaterial to the droplet, and applying energy to the droplet.

As mentioned, the first sheet may have a first hydrophobic andoleophobic surface that is positioned opposite from a second hydrophobicand oleophobic surface of the second sheet.

In general, the methods and apparatuses described herein may manipulatethe droplet(s) within the air gap by reducing the gap width (e.g., thedistance between the upper and lower sheets), which may pull the dropletinto the reduced-height region by capillary action. It may beparticularly beneficial to have the air gap may be open to atmosphericpressure, and unpressurized. This is in contrast to systems that drivethe droplet by pressure (e.g., squeezing the fluid material between thesheets), in an attempt to push the droplet, or alternatively to suck thedroplet by negative pressure.

Any of these methods may include applying the compression force againstthe first sheet using the mechanical force applicator draws the droplettowards the region of locally reduced air gap by capillary action.

In general, these methods may be part of a method of any one of nucleicacid extraction, library preparation, sequencing, and protein synthesis.For example, the step of introducing, applying and moving may be part ofa method of one or more of: nucleic acid extraction, librarypreparation, sequencing, and protein synthesis.

In general, any appropriate mechanical force applicator may be used. Forexample, a tip of the mechanical force applicator may have a roundedprofile, a circular profile, an oval profile, a rectangular profile, ora square profile. In some examples a tip of the mechanical forceapplicator comprises a roller.

Any of these methods and apparatuses may be configured to detect a lighttransmitted or reflected through the droplet. Any of these methods orapparatuses may be configured to apply a voltage to the droplet from themechanical force applicator or from a region beneath the second sheet.Any of these methods may include attracting magnetic particles suspendedwithin the droplet via a magnet within the mechanical force applicatoror a region beneath the second sheet.

In general, these methods may include mixing the droplet within the airgap. Mixing may be chaotic or gentle. For example chaotic mixing may beperformed on the droplet by a repeated application and removal of thecompression force by the mechanical force applicator, e.g., moving themechanical force applicator (or any force applicator) in the z axis,transverse to the first sheet. Alternatively or additionally, mixing thedroplet may be performed by moving the mechanical force applicatoragainst the first sheet in a plane of the first sheet, e.g., in an xand/or y axis direction.

The methods and apparatuses described herein may be configured toseparate the droplet by: applying a pinning compression force to thefirst sheet; and applying an actuation compression force to the firstsheet proximate to the pinning compression force to elongate and dividethe droplet, wherein the pinning compression force is greater than theactuation compression force. Any of these methods may include removingall or a portion of the droplet from the air gap through an opening inthe first sheet. Any of these methods may include introducing thedroplet by passing the droplet through an opening in the first sheetfrom the mechanical force applicator.

As mentioned, also described herein are methods of combining dropletsusing mechanical actuation through the elastically deformable sheet. Forexample, any of these methods may include applying a compression forceto the first sheet to deform the first sheet between two or moreseparate fluidic droplets; and releasing the compression force tocombine the two or more separate fluidic droplets into a single fluidicdroplet.

Also described herein are methods of separating droplets usingmechanical actuation through the elastically deformable sheet. Forexample, a method may include alternately applying a first compressionforce and a second compression force different than the firstcompression force to the first sheet to mix together two or moreseparate fluidic droplets within the air gap.

Also described herein are methods of mixing droplets using mechanicalactuation through the elastically deformable sheet. For example, any ofthese methods may include repeatedly applying and releasing acompression force to an outer surface of the first sheet in a region ofthe first sheet that is adjacent to two or more fluidic droplets withinthe air gap in order to mix together the two or more fluidic droplets.In addition or instead of mixing by moving the mechanical forceapplicator in the z-axis, any of these methods and apparatuses may mixby moving the mechanical force applicator against the first sheet in they or x axis (e.g., in the plane of the sheet), which may result in moregentle mixing than the more chaotic mixing resulting from moving themechanical force applicator in the z direction. Gentle mixing may beparticularly preferred when mixing long polynucleotides in order toavoid shearing.

These methods may also be used with magnetic beads or particles that maybe condensed (e.g., using a magnet, as described herein), washed, andresuspended. The methods may include any of the moving, mixing, dividingand combining of droplets. The controller may be configured to executeand control any of these steps. Also described herein are methods ofattracting, with a magnet outside of the air gap, ferrous particlessuspended within the fluidic droplet. Any of these methods may alsoinclude re-suspending one or more ferrous particles in the fluidicdroplet by applying a compression force to an outer surface of the firstsheet over or adjacent to the fluidic droplet within the air gap anddisabling a controllable magnet.

In some examples the methods described herein may include restrictingmovement of the first fluidic droplet via two or more pinning postsdisposed within the air gap. Alternatively or additionally these methodsmay include using a well to hold the droplet, particularly whenadjusting the temperature (e.g., thermal cycling). Holding the dropletin a well (and/or ‘pinning’ the droplet with one or more pinning posts)may prevent unintentional movement of the droplet during operation,including in particular when heating the droplet. Any of the methodsdescribed herein may include heating, by a heating element, the dropletrestricted by the well and/or pinning post(s). Any of these methods mayinclude heating, e.g., by a heating element (of the mechanicalmicrofluidics actuator), the fluidic droplet in the well.

In use, any of these methods and apparatuses may be used with a coatingmaterial, which may be referred to as a gloss coat (e.g., also referredto equivalently as drop gloss, gloss coating or gloss material). Thegloss coat may be a low surface-tension material (e.g., oil), and may beimmiscible with the droplet. The gloss coating material may be ahydrophobic material if the droplet is an aqueous material. The glosscoating may be applied to the droplet before it is applied into the airgap, or after it is applied to the air gap. The gloss coat may beremoved, e.g., by wicking to a material that is absorbent for the glossmaterial. The gloss coat may be particularly helpful in preventingevaporation of the droplet within the air gap.

Also described herein are methods including electroporating of cells orparticles within the droplet. For example, any of these methods mayinclude applying energy to an electrode on the plate and/or the forceapplicator to create temporary pores in cell membranes of cells withinthe fluidic droplet.

In general, the methods and apparatuses described herein may beparticularly useful for treating fluidic droplets having a variety oflow- and medium- volumes. For example, the methods described herein maybe useful for fluidic droplets having a volume of between about 10⁻¹⁵and 10⁻⁶ liters.

Also described herein are mechanical microfluidics actuator apparatuses.These microfluidic apparatuses may include: a cartridge seating surface(“seat”); a force applicator configured to contact an elasticallydeformable outer surface of a cartridge when the cartridge is seated inthe cartridge seating surface to apply a compression force to theelastically deformable surface of the cartridge; a force applicatordrive configured to move the force applicator across the deformableouter surface; and a controller coupled to control the force applicatordrive to move the force applicator relative to the deformable outersurface of the cartridge to dynamically reduce a height of an air gapwithin the cartridge to move a fluidic droplet within the air gap of thecartridge. The force applicator drive may include one or more motors formoving the force applicator in x, y and/or z. Although most of theexamples shown herein are configured to move the force applicator whilethe cartridge remains fixed (e.g., the force applicator is movedrelative to the elastically deformable sheet and air gap) in someexamples the apparatus may be configured to move the cartridge (e.g.,the elastically deformable sheet and air gap) while the force applicatorremains fixed; alternatively both the force applicator and the cartridge(e.g., the elastically deformable sheet and air gap) may move relativeto each other.

The mechanical microfluidics actuator may include one or more forceapplicators. In some example multiple force applicators may becontrolled independently (in parallel or in series). In some examples,the mechanical microfluidics actuator may switch out one type of forceapplicator for another type. Ins some examples the mechanicalmicrofluidics actuator may be configured to perform multiple concurrentactuations using multiple different force applicators.

The force applicator may have any appropriate shape, particularly shapesthat apply sufficient compression to the elastically deformable sheet soas to reduce the heigh of the air gap, while preventing or limitingdamage to the sheet. For example, a tip of the force applicator may beconfigured to have a profile comprising a circle, an oval, a rectangle,or a square. In some examples the force applicator comprises a wheel,ball point or roller.

In some examples the force applicator may be adapted to perform one ormore additional functions, in addition to applying a force against theelastically deformable sheet of air gap in order to decrease the heightof the air gap to drive movement (e.g., by 2D capillary action) of adroplet within the air gap. In some examples the tip of the forceapplicator includes a thermal output configured to control a temperatureof the tip. In some examples the tip of the force applicator includes alight source. In some examples the tip of the force applicator includesa light source, and the cartridge seating source comprises a lightsensor configured to detect light transmitted or reflected through afluidic droplet.

In some examples the force applicator includes an electrode configuredto apply a voltage (e.g., for applying electroporation). In someexamples the force applicator includes a magnet. For example, the forceapplicator may be further configured to provide a variable magneticfield strength. In some examples the force applicator comprises asonication probe configured to emit at least one of sonic and ultrasonicwaves.

Also described herein are method of moving a droplet using mechanicalactuation through the elastically deformable sheet. For example, thesemethod may include: introducing a fluidic droplet into an air gap formedbetween a first elastically deformable sheet and a second sheet, whereinthe first sheet is approximately parallel to and spaced opposite fromthe second sheet by a predetermined distance to form the air gap; andapplying a compression force against the elastically deformable sheetusing a force applicator, thereby reducing the predetermined distance inat least one region within the air gap adjacent to the fluidic dropletto move the droplet within the air gap.

Any of these methods may also include moving the force applicator acrossthe elastically deformable sheet while applying the compression force sothat the droplet follows a region of decreased distance (e.g., height)within the gap that is formed by the force applicator. A tip of theforce applicator may be configured to have a rounded profile, a circularprofile, an oval profile, a rectangular profile, or a square profile. Insome examples the tip is a roller, ball point or wheel.

Any of the methods described herein may also include controlling atemperature of a region beneath the second sheet to control atemperature of the fluidic droplet within the air gap. In some examples,the method may include controlling a temperature of the force applicatorto control a temperature of the fluidic droplet. In some examples themethod may include detecting a light transmitted or reflected throughthe fluidic droplet. The light may be part of a sensor for detecting thepresence of the droplet and/or one or more characteristics of thedroplet. The light may be emitted by a light source on the forceapplicator.

Any of these methods may include applying a voltage from the forceapplicator or from a region beneath the second sheet. The first sheetand/or the second sheet may be a dielectric material. Any of thesemethods may include attracting, via a magnet within the force applicatoror a region beneath the second sheet, ferrous particles (e.g., magneticbeads) suspended within the fluidic droplet. In some examples the methodmy include removing ferrous particles from the fluidic droplet. Themethods may include mixing the fluidic droplet via a repeatedapplication and removal of the compression force by the forceapplicator. Any of these methods may include aspirating the fluidicdroplet through an opening in the sheet using the force applicator.

In some examples the methods may include delivering the fluidic dropletinto the air gap from the force applicator. Introducing the fluidicdroplet may comprise passing the droplet through an opening in theelastically deformable sheet from the force applicator. The method mayinclude applying at least one of sonic and ultrasonic energy to thefluidic droplet from the force applicator.

Thus, described herein are methods and apparatuses (e.g., devices andsystems, including cartridges) for preparing, manipulating and/oranalyzing fluidic droplets, such as microfluidic droplets. For example,described herein are microfluidic apparatuses that may be especiallyhelpful for handling and analyzing a clinical, laboratory, biological,or chemical samples. These apparatuses may generally operate by applyinga force, such as a compression force, to an elastically deformable sheetthat at least partially covers an air gap over a subregion of the airgap, to reduce the height of the air gap in a region that is adjacent toa droplet, causing the droplet to move towards this region of reducedheight. By controlling the relative height of the air gap near thedroplet (e.g., by controlling the application of force to deform theelastically deformable sheet), a droplet may be efficiently and quicklymoved around the air gap, allowing processing of the droplet includingcombining the droplet, dividing the droplet, mixing the droplet,cooling/heating the droplet (e.g., thermocycling the droplet), usingmagnetic particles within the droplet (e.g., to bind/remove materialsfrom the droplet) and the like.

The apparatuses described herein may include two parallel hydrophobicand oleophobic sheets spaced apart by a gap of a predetermined distance.The gap may be filled with air or an immiscible fluid, with respect tothe microfluidic droplet. The microfluidic droplet may be manipulated(e.g., moved, controlled, separated, mixed, and the like) by selectivelyreducing the gap, particularly near the microfluidic droplet. In someexamples, the gap may be reduced applying a force (e.g., a compressiveforce) to one or more of the parallel sheets.

Examples described in this disclosure may be implemented as amicrofluidic device. The microfluidic device may include a cartridgethat, in turn, includes a first sheet comprising a first surface and asecond surface, wherein the first surface of the first sheet ishydrophobic and oleophobic, a second sheet comprising a first surfaceand a second surface, wherein the first surface of the second sheet ishydrophobic and oleophobic and the first surface of the first sheet isdisposed toward and separated from the first surface of the second sheetby a predetermined distance to form a gap between the first sheet andthe second sheet, and at least one input port on the first sheetconfigured to introduce a first microfluidic droplet into the gap. Themicrofluidic device may also include a controller configured selectivelyreduce the predetermined distance in one or more regions within the gapadjacent to the first microfluidic droplet, wherein the reducedpredetermined distance moves the first microfluidic droplet within thecartridge.

In some examples, the controller of the microfluidic device may beconfigured to apply a compression force to the second surface of thefirst sheet to selectively reduce the predetermined distance.

In some examples, the first sheet may be configured to deflect inresponse to compression forces and the second sheet may be configured toresist deflection in response to compression forces.

In some examples, the controller may be configured to apply a pinningcompression force to divide the first microfluidic droplet and apply anactuation compression force proximate to the pinning compression forceto elongate and form a second microfluidic droplet from the firstmicrofluidic droplet, where the pinning compression force is greaterthan the actuation compression force.

In some examples, the controller may be configured to apply acompression force to the first sheet between two or more separatemicrofluidic droplets and release the compression force to combine thetwo or more separate microfluidic droplets into a single microfluidicdroplet.

In some examples, the controller may be configured to alternately applya first compression force and a second compression force different thanthe first compression force to the first sheet to mix together two ormore separate microfluidic droplets.

In some other examples, the controller may be configured to mix togethertwo or more microfluidic droplets by repeatedly applying and releasing acompression force to the first sheet adjacent to the two or moremicrofluidic droplets.

In some examples, the controller may be configured to control a magnetto attract ferrous particles suspended within the first microfluidicdroplet.

In some other examples, the controller may be further configured tore-suspend one or more ferrous particles in the first microfluidicdroplet by applying and releasing a compression force to the firstmicrofluidic droplet and disabling a magnet.

In some examples, the first sheet may further comprise two or morepinning posts disposed on the first surface and extending into the gap,the pinning posts being configured to restrict movement of the firstmicrofluidic droplet. In such cases, the device may further include aheater disposed under the second surface opposite the two or morepinning posts.

In some examples, the cartridge may further comprise a well-disposedthrough an opening on the second sheet and configured to restrictmovement of the first microfluidic droplet. In such cases, the cartridgemay further include a heating element disposed beneath the well andconfigured to heat the first microfluidic droplet.

In some examples, the second sheet may further comprise an electrodeconfigured to create temporary pores in cell membranes of cells withinthe first microfluidic droplet.

In some other examples, the first microfluidic droplet may have a volumebetween 10⁻ ⁶ and 10⁻¹⁵ liters.

Examples described in this disclosure may be implemented as a method ofmanipulating one or more microfluidic droplets. The method may includeintroducing a first microfluidic droplet into a gap formed between afirst sheet comprising a first surface and a second surface, wherein thefirst surface of the first sheet is hydrophobic and oleophobic and asecond sheet comprising a first surface and a second surface, whereinthe first surface of the second sheet is hydrophobic and oleophobic andthe first surface of the first sheet is disposed toward and separatedfrom the first surface of the second sheet by a predetermined distanceto form the gap. The method may further include selectively reducing, bya control unit, the predetermined distance in one or more regions withinthe gap adjacent to the first microfluidic droplet to move the firstmicrofluidic droplet.

In some examples, the method may include applying a compression force tothe second surface of the first sheet to selectively reduce thepredetermined distance.

In some other examples, the first sheet may be configured to deflect inresponse to compression forces and the second sheet is configured toresist deflection in response to compression forces.

In some examples, the method may include forming a second microfluidicdroplet from the first microfluidic droplet by applying a pinningcompression force to the first sheet to divide the first microfluidicdroplet and applying an actuation compression force proximate to thepinning compression force to elongate and form the second microfluidicdroplet, where the pinning compression force is greater than theactuation compression force.

In some examples, the method may include applying a compression force tothe first sheet between two or more separate microfluidic droplets andreleasing the compression force to combine the two or more separatemicrofluidic droplets into a single microfluidic droplet.

In some examples, the method may include alternately applying a firstcompression force and a second compression force different than thefirst compression force to the first sheet to mix together two or moreseparate microfluidic droplets.

In some other examples, the method may include repeatedly applying andreleasing a compression force to the second sheet adjacent to two ormore microfluidic droplets to mix together the two or more microfluidicdroplets.

In some examples, the method may include attracting, with a controllablemagnet, ferrous particles suspended within the first microfluidicdroplet. In another example, the method may include re-suspending one ormore ferrous particles in the first microfluidic droplet by applying acompression force to the first microfluidic droplet and disabling acontrollable magnet.

In some examples, the method may include restricting the movement of thefirst microfluidic droplet via two or more pinning posts disposed on thefirst surface of the first sheet extending into the gap. Furthermore,the method may include heating, by a heating element, the firstmicrofluidic droplet restricted by the two or more pinning posts.

In some examples, the method may include restricting movement of thefirst microfluidic droplet via a well. Furthermore, the method mayinclude heating, by a heating element, the first microfluidic droplet inthe well.

In some examples, the method may include creating temporary pores, withan electrode on the second sheet, in cell membranes of cells within thefirst microfluidic droplet.

In some examples, the first microfluidic droplet may have a volumebetween 10⁻⁶ and 10⁻¹⁵ liters.

Other examples described in this disclosure may be implemented as anon-transitory computer-readable storage medium comprising instructionsthat, when executed by one or more processors of a device, cause thedevice to perform operations. The operations may include sensing a firstmicrofluidic droplet disposed in a gap between first sheet comprising afirst surface and a second surface, wherein the first surface of thefirst sheet is hydrophobic and oleophobic and a second sheet comprisinga first surface and a second surface, wherein the first surface of thesecond sheet is hydrophobic and oleophobic and the first surface of thefirst sheet is disposed toward and separated from the first surface ofthe second sheet by a predetermined distance to form the gap. Theoperation may further include selectively reducing the predetermineddistance in one or more regions within the gap adjacent to the firstmicrofluidic droplet to move the first microfluidic droplet.

Other examples described in this disclosure may be implemented as adevice that includes a first sheet comprising a first surface and asecond surface, wherein the first surface of the first sheet ishydrophobic and oleophobic, a second sheet comprising a first surfaceand a second surface, wherein the first surface of the second sheet ishydrophobic and the first surface of the first sheet is disposed towardand separated from the first surface of the second sheet by apredetermined distance to form a gap between the first sheet and thesecond sheet, and a pressure actuator coupled to the first sheet andconfigured to apply pressure to the first sheet to selectively reducethe gap between the first sheet and the second sheet.

Other examples described herein may be implemented as a device thatincludes a cartridge including a first sheet comprising a first surfaceand a second surface, wherein the first surface of the first sheet ishydrophobic and oleophobic, a second sheet comprising a first surfaceand a second surface, wherein the first surface of the second sheet ishydrophobic and oleophobic and the first surface of the first sheet isdisposed toward and separated from the first surface of the second sheetby a predetermined distance to form a gap between the first sheet andthe second sheet, and a stylus configured to contact the first sheet andselectively reduce the predetermined distance in at least one regionwithin the gap,

In some examples, the predetermined distance may be reduced by acompression force provided by the stylus. Furthermore, in some examplesthe reduced predetermined distance may cause a microfluidic droplet tomove within the gap. The tip of the stylus may have a profile of acircle, oval, rectangle, square, or a combination thereof.

In some examples, a tip of the stylus may include a temperature controldevice configured to control the temperature of a microfluidic dropletwithin the gap. In some other examples, a tip of the stylus may includea light source and the second sheet includes a light sensor configuredto detect light transmitted or reflected through a microfluidic droplet.Furthermore, the tip of the stylus may include an electrode configuredto receive a voltage sufficient to attract a microfluidic dropletthrough the first sheet. In some examples the first sheet may be adielectric material.

In some examples, the stylus of the device may include a magnetconfigured to attract ferrous particles suspended within a microfluidicdroplet disposed within the gap. The magnet may be further configured toprovide a variable magnetic field strength. to filter ferrous particlesfrom the microfluidic droplet.

Other examples described herein may be implemented as a method ofmanipulating one or more microfluidic droplets. The method may includeintroducing a microfluidic droplet into a gap formed between: a firstsheet comprising a first surface and a second surface, wherein the firstsurface of the first sheet is hydrophobic and oleophobic and a secondsheet comprising a first surface and a second surface, wherein the firstsurface of the second sheet is hydrophobic and oleophobic and the firstsurface of the first sheet is disposed toward and separated from thefirst surface of the second sheet by a predetermined distance to formthe gap. The method may further include providing, by a stylus, acompression force thereby reducing the predetermined distance in atleast one region within the gap adjacent to the microfluidic droplet.

In some examples, the reduced predetermined distance may cause themicrofluidic droplet to move within the gap. The tip of the stylus maybe configured to have a profile of a circle, oval, rectangle, square, ora combination thereof.

Some examples of the methods described herein may include controllingthe temperature of the microfluidic droplet with a temperature controldevice disposed on a tip of the stylus. Some examples of the methodsdescribed herein may include detecting light transmitted or reflectedthrough the microfluidic droplet via a light sensor. In some examples,the methods may include providing a voltage to an electrode disposed onthe stylus, wherein the voltage attracts the microfluidic dropletthrough the first sheet toward the stylus. The first sheet may be adielectric material.

In some examples, the methods may include attracting, via a magnetwithin the stylus, ferrous particles suspended within the microfluidicdroplet. Furthermore, the method may include filtering ferrous particlesfrom the microfluidic droplet. The methods may include dispersingparticles within the microfluidic droplet via a repeated application andremoval of a compression force by the stylus.

In some examples, the methods may further include aspirating, by thestylus, the microfluidic droplet through a first septa in the firstsheet and receiving, by a lumen in the stylus coupled to the firstsepta, the microfluidic droplet. In some examples, methods may furtherinclude injecting, by the stylus, the microfluidic droplet into a secondsepta different than the first septa. In some further examples, themethod may include controlling, by a temperature control element in thestylus, the temperature of the microfluidic droplet. In some examples,the method may include detecting, within the stylus, light transmittedor reflected by the microfluidic droplet. In some further examples, themethod may include providing, within the stylus, at least one of sonicand ultrasonic waves to the microfluidic droplet.

Other examples described herein may be implemented as a non-transitorycomputer-readable storage medium comprising instructions that, whenexecuted by one or more processors of a device, may cause the device toperform operations comprising: sensing a first microfluidic dropletdisposed in a gap between first sheet comprising a first surface and asecond surface, wherein the first surface of the first sheet ishydrophobic and oleophobic and a second sheet comprising a first surfaceand a second surface, wherein the first surface of the second sheet ishydrophobic and oleophobic and the first surface of the first sheet isdisposed toward and separated from the first surface of the second sheetby a predetermined distance to form the gap and providing, by a stylus,a compression force thereby reducing the predetermined distance in atleast one region within the gap adjacent to the microfluidic droplet.

Any of the methods and apparatuses described herein may be adapted toperform sequency by synthesis. For example, described herein are methodsof sequencing by synthesis using mechanical compression, the methodcomprising: sequentially moving each droplet of a series of fluidicdroplets within a cartridge, wherein the cartridge comprises an air gapformed between a first sheet having a first surface that is hydrophobicand oleophobic and a second sheet having a second surface that ishydrophobic and oleophobic, wherein the first sheet and the second sheetare secured opposite each other at a predetermined spacing relative toeach other with the air gap therebetween, further comprising a pluralityof clusters of polynucleotides on the first sheet or the second sheet,wherein moving each droplet comprises moving one or more mechanicalforce applicators against the first sheet to elastically deform thefirst sheet and create a region of reduced spacing between the first andsecond sheets so that each droplet follows the region of reduced spacingover the plurality of clusters of polynucleotides, wherein the series offluidic droplets includes: a nucleotide polymerase reaction mix droplet,a first wash buffer droplet, a dye/terminator cleavage mix droplet, anda second wash buffer droplet; and imaging the plurality of clusters ofpolynucleotides to detect addition or hybridization of a nucleotide toclusters of the plurality of clusters of polynucleotides.

Any of these methods may include repeating the steps of sequentiallymoving each droplet and imaging the plurality of clusters to generatesequence data for the plurality of clusters of polynucleotides. Thesemethods may also include introducing the series of fluidic droplets intothe air gap. For example, sequentially moving each droplet may comprisemoving each droplet as it is introduced into the air gap. In any ofthese methods, imaging may comprise imaging the plurality of clusters ofpolynucleotides after moving the first wash buffer droplet over theplurality of clusters of polynucleotides. The plurality of clusters ofpolynucleotides may be arranged in a patterned or, in some examples, anun-patterned arrangement.

In any of these examples, moving the one or more mechanical forceapplicators against the first sheet to elastically deform the firstsheet comprises moving one or more styluses against the first sheet.

Any of these methods may include mixing. For example, the method mayinclude chaotically mixing by repeatedly applying and releasing acompression force to the first sheet over the plurality of clusters ofpolynucleotides to mix one droplet of the series of fluidic dropletsover the plurality of clusters of polynucleotides. Alternatively oradditionally, the methods may include gently mixing by moving themechanical force applicator against the first sheet in a plane of thefirst sheet, e.g., in an x and/or y axis direction.

Sequentially moving each droplet of the series of fluidic droplets maycomprise removing each droplet of the series of fluidic droplets fromthe air gap after it been moved over the plurality of clusters ofpolynucleotides.

The method described herein may be used with very low volume droplets.For example, the droplets of the series of fluidic droplets has a volumebetween 10⁻⁶ and 10⁻¹⁵ liters (e.g., microliter or less).

In general, in an of these methods, the air gap may be open toatmospheric pressure and unpressurized. Thus, the droplet is not movedby pressure, but instead by the change in gap width (also referred to asgap height), resulting in a capillary force pulling the droplet.

Any of these methods may include hybridizing a library ofpolynucleotides to either the first sheet or the second sheet in the airgap to form the clusters of polynucleotides.

In some examples a method of sequencing by synthesis using mechanicalcompression may include: sequentially moving each droplet of a series offluidic droplets within a cartridge, wherein the cartridge comprises anair gap formed between a first sheet having a first surface that ishydrophobic and oleophobic and a second sheet having a second surfacethat is hydrophobic and oleophobic, wherein the first sheet and thesecond sheet are secured opposite each other at a predetermined spacingrelative to each other with the air gap therebetween, further comprisinga plurality of clusters of polynucleotides on the second sheet, whereinmoving each droplet comprises moving one or more mechanical forceapplicators against the first sheet to elastically deform the firstsheet and create a region of reduced spacing between the first andsecond sheets so that each droplet follows the region of reduced spacingover the plurality of clusters of polynucleotides, wherein the series offluidic droplets includes: a nucleotide polymerase reaction mix droplet,a first wash buffer droplet, a dye/terminator cleavage mix droplet, anda second wash buffer droplet; imaging the plurality of clusters ofpolynucleotides to detect addition or hybridization of a nucleotide toclusters of the plurality of clusters of polynucleotides; and repeatingthe steps of sequentially moving each droplet and imaging the pluralityof clusters to generate sequence data for the plurality of clusters ofpolynucleotides.

A method of sequencing by synthesis using mechanical compression mayinclude: sequentially introducing a series of fluidic droplets into anair gap formed between: a first sheet having a first surface that ishydrophobic and oleophobic; and a second sheet having a second surfacethat is hydrophobic and oleophobic, wherein the first sheet and thesecond sheet are secured opposite at a predetermined spacing relative toeach other with the air gap therebetween, further comprising a pluralityof clusters of polynucleotides on the second sheet; sequentially movingeach droplet of the series of fluidic droplets over the plurality ofclusters of polynucleotides by moving one or more mechanical forceapplicators against the first sheet to elastically deform the firstsheet and create a region of reduced spacing between the first andsecond sheets so that each droplet follows the region of reduced spacingover the plurality of clusters of polynucleotides, wherein the series offluidic droplets includes: a nucleotide polymerase reaction mix droplet,a first wash buffer droplet, a dye/terminator cleavage mix droplet, anda second wash buffer droplet; imaging the plurality of clusters ofpolynucleotides to detect addition or hybridization of a nucleotide toclusters of the plurality of clusters of polynucleotides; and repeatingthe steps of sequentially introducing the series of fluidic droplets,moving each droplet and imaging the plurality of clusters.

All of the methods and apparatuses described herein, in any combination,are herein contemplated and can be used to achieve the benefits asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the methods andapparatuses described herein will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,and the accompanying drawings of which:

FIGS. 1A-1C show a portion of a microfluidic apparatus (e.g., a portionof a mechanical microfluidics actuator or cartridge for use in amechanical microfluidics actuator).

FIG. 2 is a flowchart showing an example operation for manipulating amicrofluidic droplet.

FIGS. 3A-3C show a portion of another microfluidic apparatus (e.g.,mechanical microfluidics actuator).

FIG. 4 is a flowchart showing an example operation for separating amicrofluidic droplet into two or more microfluidic droplets.

FIGS. 5A-5C show a portion of another microfluidic apparatus.

FIG. 6 is a flowchart showing an example operation for mixing amicrofluidic droplet.

FIGS. 7A-7C show a portion of another microfluidic apparatus.

FIG. 8 is a flowchart showing an example operation for removingsuspended ferrous particles from a microfluidic droplet.

FIGS. 9A-9B show a portion of another microfluidic apparatus.

FIG. 10 is a flowchart showing an example operation for dispersingparticles in a microfluidic droplet.

FIGS. 11A-11C show a portion of another microfluidic apparatus.

FIG. 12 is a flowchart showing an example operation for manipulating amicrofluidic droplet in conjunction with pinning posts.

FIGS. 13A-13E show a portion of another microfluidic apparatus.

FIG. 14 is a flowchart showing an example operation for manipulating amicrofluidic droplet in conjunction with a well.

FIGS. 15A-15C show a portion of another microfluidic apparatus.

FIG. 16 is a flowchart showing an example operation for providingelectroporation.

FIG. 17 shows an example microfluidic apparatus (e.g., a mechanicalmicrofluidics actuator).

FIG. 18A shows an example of a portion of a microfluidic apparatus asdescribed herein.

FIG. 18B shows possible associated end profiles of a stylus such as thatshown in FIG. 18A.

FIGS. 19A and 19B show a portion of another microfluidic apparatus.

FIG. 20 is a flowchart showing an example operation for manipulating amicrofluidic droplet.

FIGS. 21A and 21B show a portion of another microfluidic apparatus.

FIG. 22 is a flowchart showing an example operation for manipulating amicrofluidic droplet.

FIGS. 23A-23C show a portion of another microfluidic apparatus.

FIG. 24 is a flowchart showing an example operation for removingsuspended ferrous particles from a microfluidic droplet.

FIGS. 25A and 25B show a portion of another microfluidic apparatus.

FIG. 26 is a flowchart showing an example operation for dispersingparticles in a microfluidic droplet.

FIGS. 27A-27C show a portion of another microfluidic apparatus.

FIG. 28 is a flowchart showing an example operation for aspiratingliquids in a microfluidic apparatus.

FIGS. 29A-29C shows a portion of another microfluidic apparatus.

FIG. 30 is a flowchart showing an example operation for manipulatingliquids in a microfluidic apparatus.

FIGS. 31A-31C shows a portion of another microfluidic apparatus.

FIG. 32 is a flowchart showing an example operation for thermallytreating liquids in a microfluidic apparatus.

FIG. 33 shows a portion of another microfluidic apparatus.

FIGS. 34A-34C show a portion of another microfluidic apparatus.

FIG. 35 is a flowchart showing an example operation for performing areaction measurement.

FIG. 36 shows a portion of another microfluidic apparatus.

FIGS. 37A-37C show a portion of another microfluidic apparatus.

FIG. 38 is a flowchart showing an example operation for performing asonic treatment.

FIG. 39 shows a block diagram of a device that may be one example of amicrofluidic apparatus (e.g., mechanical microfluidics actuator) asdescribed herein.

FIGS. 40A-40D illustrate one examples cartridges for use with amechanical microfluidics actuator. FIG. 40A shows an example of acartridge having three lanes. FIG. 40B shows an example of a cartridgehaving eight lanes. FIG. 40C shows an example of a portion of thecartridge of FIG. 40A; FIG. 40D shows an enlarged view of the sectionalview of FIG. 40C.

FIG. 41 is an exploded view of an example of another example of acartridge.

FIGS. 42A and 42B show partial sectional views of a portion ofcartridge.

FIG. 43A shows a section view through another example of a cartridge.

FIGS. 43B and 43C illustrate the cartridge of FIG. 43A seated in amechanical microfluidics actuator. FIG. 43C shows the cartridge of FIG.43B with a droplet within the air gap of the cartridge.

FIG. 43D shows an example of a cartridge seated in an actuator assemblywith three parallel lane being concurrently actuated by a roller stylus.

FIG. 43E shows an example of an eight-lane cartridge as describedherein.

FIGS. 44A-44E illustrate examples of different seating regions of amechanical microfluidics actuator coupled with a cartridge as describedherein. FIG. 44A shows an example of a cartridge coupled to a mechanicalmicrofluidics actuator having a seating region including a recessedseating surface. FIG. 44B shows a mechanical microfluidics actuatorhaving a seating region with a raised section. FIG. 44C shows an exampleof a mechanical microfluidics actuator having a thermally-conductivecylindrical rail (forming a rail region) projecting from the seatingregion. FIG. 44D shows an example of a mechanical microfluidics actuatorhaving a thermally-conductive flat platform rail (e.g., rail region)projecting from the seating region. FIG. 44E shows an example of amechanical microfluidics actuator having a thermally-conductive domedplatform rail (rail region) projecting from the seating region.

FIGS. 45A-45C illustrate an example of a seating region of a mechanicalmicrofluidics actuator. FIG. 45A shows the seating region; FIG. 45Bshows the seating region made partially transparent; FIG. 45C shows theseating region with a cartridge coupled thereto.

FIGS. 45D and 45E illustrate enlarged sections through the seatingregion example of FIGS. 45A-45C.

FIG. 46 schematically illustrates one example of a seating portion of acartridge coupled to a seating region.

FIG. 47 illustrates a method of transferring a small volume of fluidusing a solid (e.g., non-vacuum/non-pipette) technique.

FIG. 48 schematically illustrates an example of a mechanicalmicrofluidics actuator as described herein.

FIG. 49 illustrates an example of an internal region (showing a row ofmagnetic elements) of a mechanical microfluidics actuator .

FIG. 50 shows one example of a mechanical microfluidics actuator.

FIG. 51 illustrates an example of a multiplexed apparatus includingmultiple mechanical microfluidics actuators.

FIGS. 52A-52B show a schematic illustration of one example of anassembly forming a mechanical microfluidics actuator.

FIGS. 53A-53C illustrate a method of operating an apparatus as describedherein.

FIG. 54 schematically illustrates a sequencing by synthesis (SBS) methodthat may be implemented by the techniques described herein.

FIGS. 55A-55B illustrate an example of introducing sequencing primers toDNA template in a cartridge (e.g., flow cell) having discrete clusters,which may be part of an SBN method performed using the methods andapparatuses described herein.

FIGS. 56A-56E schematically illustrate the delivery of SBS reagentsusing the methods and apparatuses described herein.

FIG. 57 illustrate a cartridge as described herein.

FIG. 58 schematically illustrates a method for polynucleotide extractionfrom a tissue sample (e.g., blood) that may be performed using themethods and apparatuses described herein.

FIG. 59 schematically illustrates a method of performing an RNAseqworkflow using the methods and apparatuses described herein.

FIG. 60 schematically illustrates a method for Twist exome targetenrichment fast hybridization that may be performed using the methodsand apparatuses described herein.

FIG. 61 schematically illustrates a workflow for performing Amplisequ (2primer pools) using the methods and apparatuses described herein.

FIGS. 62A-62J illustrate loading and unloading of droplets within acartridge as described herein.

FIG. 63 illustrates evaporation prevention using the methods andapparatuses described herein.

DETAILED DESCRIPTION

A microfluidic apparatus (e.g., device, system or the like) forcontrolled liquid manipulation may include a two-dimensional (planar)fluidic chamber. The chamber may include a first sheet and a secondsheet separated by a gap therebetween. The gap may separate the firstand second sheets by any feasible distance. The first and second sheetsmay be hydrophobic or may include hydrophobic coatings. In some examplesthe first and second sheet are hydrophobic and oleophobic and/or includea hydrophobic and oleophobic coating.

Microfluidic droplets may be manipulated through mechanical manipulationthat applies forces directly or indirectly to the first sheet or secondsheet selectively reducing the gap. This process may sometimes bereferred to as mechanical actuation on the surface (MAOS), alsodescribed as the use of mechanical compression to change the capillaryforce. The applied forces, which may include compressive forces, may beapplied near or adjacent to droplets within the gap. In some aspects,reducing the gap may cause the droplets to move, separate, combine, mix,incubate, or the like.

In some examples, the forces may be applied by a stylus. The stylus mayinclude an electrode and/or a controllable magnet. The microfluidicdroplets may be manipulated with a combination of pressure, exerted bythe stylus, in conjunction with a voltage provided by the electrodeand/or a magnetic field provided by the magnet.

FIG. 1A shows a portion of a microfluidic device 100. Any of the devicesdescribed herein may be implemented in part or in whole as a system orany other feasible apparatus. The microfluidic device 100 may include afirst sheet 110 and a second sheet 120 separated by a gap 130. In someexamples, the gap 130 may generally be filled with air. In someexamples, the microfluidic device 100 may be a cartridge that may beselectively coupled to a control unit or base station. As shown, thefirst sheet 110 may be a “top” sheet and the second sheet 120 may be a“bottom” sheet. That is, the first sheet 110 may be higher or “above”the second sheet 120. The second sheet 120 may be closer to the groundthan the first sheet 110. In other examples, the second sheet 120 may beabove the first sheet 110.

The first sheet 110 and the second sheet 120 may form a planar structurethat occupies any feasible area. The first sheet 110 and the secondsheet 120 are shown in an initial position. In the initial position, thefirst sheet 110 and the second sheet 120 are relatively parallel to eachother separated by a distance associated with and/or determined by thegap 130.

Each sheet may include two surfaces. For example, the first sheet 110may include a first surface 111 and a second surface 112 and the secondsheet 120 may include a first surface 121 and a second surface 122. Forease of description, the first surfaces 111 and 121 may be disposedtoward the gap 130, while the second surfaces 112 and 122 may bedisposed on opposite sides of the first sheet 110 and the second sheet120, respectively. In other words, the second surfaces 121 and 122 maybe disposed away from the gap 130.

The first surfaces 111 and 121 may be hydrophobic (water repelling). Insome examples, the first and second sheets 110 and 120 (and thus thefirst surfaces 111 and 121) may be formed from a hydrophobic andoleophobic material. In some other examples, the first surfaces 111 and121 may be a hydrophobic and oleophobic coating or layer applied uponthe first and second sheets 110 and 120, respectively.

A droplet 140 may be introduced into the gap 130. In some cases, thedroplet 140 may be introduced in the gap 130 through a port or opening(not shown) on the first sheet 110 and/or the second sheet 120. Thedroplet 140 may be mechanically manipulated by selectively reducing thegap near the droplet 140. In some examples, one or more of the sheets110 and 120 may be flexible. Flexible sheets may deflect in response toone or more forces. For example, the first sheet 110 may be flexible andthe second sheet 120 may be rigid or semi-rigid. Rigid or semi-rigidsheets may resist deflection in response to one or more forces. In otherexamples, the second sheet 120 may be flexible and the first sheet 110may be rigid or semi-rigid. In still other examples, both the firstsheet 110 and the second sheet 120 may be flexible. As used herein, theterm flexible may describe any material that may flex, deform, bend,move, or the like.

The droplet 140 may have a predetermined volume. In some cases, thedroplet 140 may be a microfluidic droplet having a volume of the droplet140 may be between 10⁻⁶ and 10⁻¹⁵ liters, although in some examples thevolume of the droplet 140 can have any other feasible volume. The gap130 may be determined, at least in part, by the volume of the droplet140. In other words, the gap 130 may be chosen or selected such that thedroplet 140 (e.g., the volume of the droplet 140) can touch both thefirst and second sheets 110 and 120.

FIG. 1B shows another view of the microfluidic device 100. In this view,the first sheet 110 may be deflected by a compression force near oradjacent to one side or end of the droplet 140. The compression forcecreates a reduced gap 132 between the first sheet 110 and the secondsheet 120 toward the end or side of the droplet 140. As the reduced gap132 is formed, the droplet 140 may deform asymmetrically and be drawntoward the reduced gap 132. In some cases, the droplet movement may becaused by differential capillary action and/or a differential pressuregradient within the droplet 140. The compression force may be providedby any feasible means. For example, an array of electro-mechanical,mechanical, and/or pneumatic actuators may be disposed next to the firstsheet 110 and/or the second sheet 120 to selectively provide acompression force to form the reduced gap 132. In another example, thecompression force may be provided by a stylus that may contact the firstsheet 110 and/or the second sheet 120.

In some examples, the microfluidic device 100 may include one or moreoptical sensors (not shown). The one or more optical sensors may detectthe presence and/or position of the droplet 140. In this manner, datafrom the optical sensors may be used to assist the application of acompression force near or adjacent to the droplet 140.

FIG. 1C shows another view of the microfluidic device 100. In this view,the compression force on the first sheet 110 has been removed or reducedand the first sheet 110 and the second sheet 120 has returned to aninitial position (as shown in FIG. 1A). The gap 130 may be similar tothe gap 130 of FIG. 1A. The droplet 140 is shown in a second positionhaving moved in response to the compression force described with respectto FIG. 1B.

Thus, in the manner described within FIGS. 1A-1C, any droplet may bemanipulated to any area within the microfluidic device 100 by reducingthe gap near one end of the droplet. This method advantageously avoidsthe generation and control of high voltages as well as the need for aplurality of electrodes that are associated with conventionalmicrofluidic devices. The compression force described herein may beprovided by any feasible source. For example, mechanical levers, balls,rollers, or the like may apply the compression force to at least one ofthe first or second sheets 110 and 120, respectively. In some examples,the compression force may be computer or processor controlled. Thus themanipulation of the droplet 140 may be computer and/or processorcontrolled.

FIG. 2 is a flowchart showing an example operation 200 for manipulatinga microfluidic droplet. Some examples may perform the operationsdescribed herein with additional operations, fewer operations,operations in a different order, operations in parallel, and someoperations differently. The operation 200 is described below withrespect to the microfluidic device 100 of FIGS. 1A-1C, however, theoperation 200 may be performed by any other suitable system or device.

The operation 200 begins in block 202 as a microfluidic droplet isintroduced into a gap between two hydrophobic and oleophobic sheets. Forexample, the droplet 140 may be loaded into the gap 130 between thefirst sheet 110 and the second sheet 120 of the microfluidic device 100.The gap 130 may be an initial separation distance between the firstsheet 110 and the second sheet 120. The first sheet 110 and the secondsheet 120 may be hydrophobic and oleophobic or include surfaces coveredwith a hydrophobic and oleophobic layer. The droplet 140 may be placedin an initial position. The gap 130 may be filled with any feasible gas,such as air. In some examples, the gap 130 may be filled with animmiscible (with respect to the droplet 140) fluid. The presence and/orlocation of the droplet 140 may be determined with an optical sensor(not shown). Example optical sensors may include one or more digitalcameras, an array of visible and/or invisible light detectors, or thelike. Thus, an optical sensor may determine when the droplet 140 isintroduced into the gap 130

Next, in block 204 the distance between the two hydrophobic andoleophobic sheets is selectively reduced, via mechanical actuation, nearthe microfluidic droplet. The reduced distance may result in a reducedgap 132 between the first sheet 110 and the second sheet 120. In someexamples, a compression force may be provided to the sheet 110 adjacentto (e.g., next to) one side of the droplet 140. The compression forcemay reduce the gap 130 causing the droplet 140 to move toward thecompression force. In some examples, the compression force may deformone end of the droplet 140.

Next, in block 206 the distance between the two hydrophobic andoleophobic sheets is restored. For example, the compression forceapplied in block 204 may be removed allowing the first sheet 110 and/orthe second sheet 120 to return to its initial separation distance. Insome examples, the distance between first sheet 110 and second sheet 120is returned to a distance similar to the initial separation distance ofblock 202. The microfluidic droplet may come to rest at a differentposition having moved from the initial position of block 202.

The steps of blocks 202-206 may be repeated any number of times tomanipulate one or more droplets to any arbitrary location in the gap 130of the microfluidic device 100. In some examples, different compressionforces (e.g., different force amplitudes) may be applied to the dropletto perform different manipulations.

FIG. 3A shows a portion of another microfluidic device 300. Themicrofluidic device 300 may include a first sheet 310, a second sheet320, and a gap 330 that may be examples of the first sheet 110, thesecond sheet 120 and the gap 130 of FIGS. 1 . A source droplet 340(which may be similar to the droplet 140) may be introduced into the gap330 as described above with respect to FIGS. 1A and 2 .

A pinning compression force may be applied to the source droplet 340.Although shown as being applied to the first sheet 310, in otherexamples the pinning compression force may be applied to the secondsheet 320. As shown, the pinning compression force may be appliedapproximately toward the center or middle of the source droplet 340. Thepinning compression force may be provided by any technical feasibledevice or operation. The pinning compression force may begin to divideor separate the source droplet 340 into two droplets.

FIG. 3B shows another view of the microfluidic device 300. As shown, anactuation compression force may be applied to the source droplet 340.The actuation compression force may be less than the pinning compressionforce applied in FIG. 3A. The actuation compression force may be appliedat the same time (coincident with) or after applying the pinningcompression force. The actuation compression force may separate andguide a satellite droplet 341 away from the source droplet 340. In someexamples, the pinning compression force may cause the first sheet 310 tocontact the second sheet 320 helping separate the satellite droplet 341from the source droplet 340.

FIG. 3C shows another view of the microfluidic device 300. As shown, thepinning and actuation forces have been removed or reduced allowing thefirst sheet 310 and/or the second sheet 320 to return to their initialpositions, such as the initial positions shown in FIG. 3A. The sourcedroplet 340 is shown separated from the satellite droplet 341.

FIG. 4 is a flowchart showing an example operation 400 for separating amicrofluidic droplet into two or more microfluidic droplets. Theoperation 400 is described below with respect to microfluidic device 300of FIGS. 3A-3C, however, the operation 400 may be performed by any othersuitable system or device.

The operation 400 begins in block 402 as a pinning compression force isapplied, via mechanical actuation, to a source microfluidic droplet. Thesource microfluidic droplet (which may be similar to the sourcemicrofluidic droplet 340) may have previously been introduced into a gapbetween the first sheet 310 and the second sheet 320, for example asdescribed above with respect to FIGS. 1A and 2 . In some examples, thepinning compression force may begin to divide or separate the sourcemicrofluidic droplet into two or more droplets. The pinning compressionforce may be a mechanical actuation force provided to the first sheet310 and/or the second sheet 320 and may be provided by any feasiblemeans. In some cases, the pinning compression force may be appliedtoward the middle or center of the source microfluidic droplet. In someexamples, the pinning compression force may cause the first sheet 310 tocontact the second sheet 320.

Next, in block 404, an actuation compression force is applied, viamechanical actuation, to the source microfluidic droplet. The actuationforce may be applied to separate, direct, and/or steer a satellitemicrofluidic droplet away from the source microfluidic droplet. Theactuation compression force may be another mechanical actuation forcethat, in this instance, is less in force than the pinning compressionforce. The actuation compression force may be applied coincident with,or after the pinning compression force is applied.

In block 406, the pinning compression force and the actuationcompression force is removed or reduced. In the absence of thecompression forces, the first sheet 310 and the second sheet 320 mayreturn to associated initial positions, such as depicted in FIG. 3C. Asthe pinning and actuation compression forces are removed or reduced, thesatellite droplet may remain separated from the source droplet.

In some examples, two or more separate droplets may be merged togetherusing a compression force. One such example is described in conjunctionwith FIGS. 5A-5C and 6 . Droplets of different sizes or the same sizemay be merged. For example, 250nL-80µL volumes may be robustly actuatedand merged with an equal volume, larger or smaller volume that alreadyexists in the air gap.

FIG. 5A shows a portion of another microfluidic device 500. As shown inFIG. 5A, the microfluidic device 500 may include a first sheet 510, asecond sheet 520, and a gap 530 that may be other examples of the firstsheet 110, the second sheet 120 and the gap 130 of FIG. 1A. A firstdroplet 540 and a second droplet 541 (which may be similar to thedroplet 140 of FIG. 1A or the source droplet 340 and satellite droplet341 of FIG. 3C) may be introduced into the gap 530 as described abovewith respect to FIGS. 1A-1C, 2, 3A-3C, and 4 .

A compression force may be applied to the first sheet 510 and/or thesecond sheet 520. In some examples, the compression force may be appliedbetween the first droplet 540 and the second droplet 541 causing therespective droplets to deform, move, and in some cases, combine. Thecompression force may be a mechanical actuation force as describedherein.

FIG. 5B, shows another view of the microfluidic device 500. Thecompression force is removed or reduced thereby allowing the first sheet510 and/or the second sheet 520 to return to initial (uncompressed)positions. As shown, the first droplet 540 and the second droplet 541may be combined into a merged droplet 542. Although combined into asingle droplet, the individual droplets, or components within the firstdroplet 540 and the second droplet 541 may not be well-mixed within themerged droplet 542.

FIG. 5C shows another view of the microfluidic device 500. A compressionforce may be repeatedly applied and released to and from the first sheet510 and/or the second sheet 520 to mix the contents of the mergeddroplet 542. The repeated application of the compression force mayrepeatedly cause the merged droplet 542 to deform and recover therebycausing the contents of the merged droplet 542 to mix. In some examples,the compression/relaxation cycle, caused by the application and releaseof the compression force, may be repeated for a predetermined number oftimes to mix the contents of the merged droplet 542.

FIG. 6 is a flowchart showing an example operation 600 for mixing amicrofluidic droplet. The operation 600 is described with respect to themicrofluidic device 500 of FIGS. 5A-5C, however the operation 600 may beperformed by any other suitable system or device. The operation 600begins in block 602 as a compression force is applied, via mechanicalactuation, to the first sheet 510 and/or the second sheet 520 betweenfirst and second microfluidic droplets 540 and 541. The compressionforce may be a mechanical actuation force provided by any feasiblemeans. The compression force may cause the first and second microfluidicdroplets 540 and 541 to move toward each other and, in some cases,combine into a single (merged) microfluidic droplet 542.

In block 604, the compression force is reduced or removed from the firstsheet 510 and/or the second sheet 520. In some examples, reducing orremoving the compression force may allow the first sheet 510 and/or thesecond sheet 520 to return to an initial position.

In some cases, additional agitation of the merged microfluidic droplet542 may be desired to provide additional mixing. To provide theadditional agitation, a compression force may be repeatedly applied andremoved (or reduced) for a predetermined number of cycles. Therefore, inblock 606, the number of completed compression cycles is determined. Acompleted compression cycle may include the application and removal orreduction of a compression force. If the number of compression cycles isless than a predetermined number, then the operation may return to block602. On the other hand, if the number of compression cycles is greaterthan or equal to the predetermined number, then the operation 600 mayend.

In some examples, ferrous particles may be suspended within amicrofluidic droplet to assist with processing or assaying. After one ormore processing steps have been completed, the ferrous particles may beremoved from the droplet for further processing.

FIG. 7A shows a portion of another microfluidic device 700. Themicrofluidic device 700 may include a first sheet 710, a second sheet720 and a gap 730 which may be examples of the first sheet 110, thesecond sheet 120, and the gap 130 of FIGS. 1 . A droplet 740 may includeone or more ferrous particles that 741 that may be suspended within thedroplet 740. The microfluidic device 700 may also include a magnet 750(not shown).

FIG. 7B shows another view of the microfluidic device 700. The magnet750 is activated or enabled. For example, the magnet 750 may be anelectromagnet that may be enabled through an application of power. Inanother example, the magnet 750 may be a permanent magnet that may bemoved toward the droplet 740. In addition, a compression force may beapplied to the first sheet 710 and/or the second sheet 720 toward oneside of the droplet 740. The compression force may be applied at or nearthe same time as the magnet 750 is enabled or moved.

The magnet 750 may cause the ferrous particles 741 to collect into oneor more ferrous beads 742. Thus, the ferrous particles 741 may come outof suspension from the droplet 740. In addition, the compression forcemay cause the droplet 740 to move away from the magnet 750. The dropletmovement may, in some cases, filter or remove the ferrous particles fromthe droplet 740.

FIG. 7C shows another view of the microfluidic device 700. Thecompression force is removed or reduced and the first sheet 710 and thesecond sheet 720 return to initial positions. The compression forceapplied in FIG. 7B and removed or reduced in FIG. 7C may cause thedroplet 740 to move away from the magnet 750. Since the magnet 750 mayattract and/or limit the movement of the ferrous bead 742, dropletmovement may filter the ferrous material from the droplet 740.

FIG. 8 is a flowchart showing an example operation 800 for removingsuspended ferrous particles from a microfluidic droplet. The operation800 is described with respect to the microfluidic device 700 of FIGS.7A-7C, however the operation 800 may be performed by any other suitablesystem or device.

The operation 800 begins in block 802 as a microfluidic droplet withsuspended ferrous particles is moved via mechanical manipulation to aregion proximate to a magnet. For example, a microfluidic droplet 740may be moved near a magnet 750. In some examples, the droplet 740 may bemoved through the application of forces to one or more sheets asdescribed herein.

Next, in block 804 a magnet is enabled. In some cases this step may beoptional as indicated with dashed lines in FIG. 8 . In some examples,the magnet 750 may be a permanent and stationary magnet. In some otherexamples, the magnet 750 may be enabled by moving the magnet 750 towardthe microfluidic droplet 740 or the magnet 750 may be an electromagnetand may receive power. The magnet 750 may cause ferrous particles tofall out of suspension and collect toward the magnet 750. In someexamples, the ferrous particles 741 may be collected into one or moreferrous beads 742.

Next, in block 806, the microfluidic droplet 740 may be moved away fromthe magnet 750 through mechanical actuation. For example, a compressiveforce may be applied to first or second sheets 710 or 720 to move themicrofluidic droplet 740 away from the magnet 750. Moving themicrofluidic droplet 740 away from the magnet may filter the ferrousmaterial from the microfluidic droplet 740.

In some examples, ferrous material may be placed back into suspensionwithin a droplet through mechanical actuation. One such example isdescribed below in conjunction with FIGS. 9-10 .

FIG. 9A shows a portion of another microfluidic device 900. Themicrofluidic device 900 may include a first sheet 910, a second sheet920, and a gap 930. The first sheet 910, the second sheet 920, and thegap 930 may be examples of the first sheet 110, the second sheet 120,and the gap 130 of FIG. 1A.

A droplet 940 may include non-dispersed ferrous or non-ferrousparticles. A compression force may be applied to the first sheet 910and/or the second sheet 920 (not shown) that can compress or deform thedroplet 940. In some cases, a compression force may be applied to thecenter or middle of the droplet 940.

FIG. 9B shows another view of the microfluidic device 900. Thecompression force may be removed or reduced from the first sheet 910and/or the second sheet 920. Removal or release of the compression forcemay allow the droplet 940 to return to a non-compressed or non-deformedstate. Transitioning from a compressed to a non-compressed state (orvice-versa) may cause one or more ferrous or non-ferrous particles 941within the droplet 940 to become suspended. In some cases, thecompression force may be applied and/or removed quickly or abruptly.Sudden application and/or removal of compression forces may assist indispersing ferrous and non-ferrous particles 941 throughout the droplet940. In some cases, the compression force may be repeatedly applied andremoved to disperse particles more evenly.

FIG. 10 is a flowchart showing an example operation 1000 for dispersingparticles in a microfluidic droplet. The operation 1000 is describedbelow with respect to the microfluidic device 900 of FIGS. 9A-9B,however the operation 1000 may be performed by any other suitable systemor device.

The operation 1000 begins in block 1002 where a compression force isapplied via mechanical actuation, to a microfluidic droplet thatincludes ferrous and/or non-ferrous particles 941 that are to besuspended. The compression force may be provided by a mechanicalactuation force that may be applied to the first sheet 910 and/or thesecond sheet 920 and also to the droplet 940. The compression force maycause the droplet 940 to deform or spread.

Next, in block 1004 the compression force may be released or reduced tosuspend the ferrous and/or non-ferrous particles 941 in the droplet 940.The removal or reduction of the compression force may cause themicrofluidic droplet 940 to return to a spherical or quasi-sphericalshape that causes ferrous or non-ferrous particles to become at leastpartially suspended within the droplet 940.

In some cases, additional agitation of the microfluidic droplet may bedesired to enhance the distribution of the particles in the microfluidicdroplet 940. To provide the additional agitation, a compression forcemay be repeatedly applied and removed (or reduced) for a predeterminednumber of cycles. Therefore, in block 1006, the number of completedcompression cycles is determined. A completed compression cycle mayinclude the application and removal or reduction of a compression force.If the number of compression cycles is less than a predetermined number,then the operation 1000 may return to block 1002. On the other hand, ifthe number of compression cycles is greater than or equal to thepredetermined number, then the operation 1000 may end.

In some examples, heating of a droplet may be desired as part of dropletanalysis or assay. However, a droplet may move during a heatingoperation and may not remain centered or positioned over a heatingelement. In some cases, pinning posts may be used to control theposition of the droplet.

FIG. 11A shows a portion of another microfluidic device 1100. As shownin FIG. 11A, the microfluidic device 1100 may include a first sheet1110, a second sheet 1120, a gap 1130, and a heater 1140. The firstsheet 1110, the second sheet 1120, and the gap 1130 may be examples ofthe first sheet 110, the second sheet 120, and the gap 130 of FIG. 1A.The first sheet 1110 may include one or more pinning posts 1150 attachedto a first surface 1111 of the first sheet 1110. In some examples, thepinning posts 1150 may be attached to the second sheet 1120.

The second sheet 1120 may include a first surface 1121 and a secondsurface 1122. The first surface 1121 may be disposed toward (e.g.,adjacent to) the gap 1130. As shown, the heater 1140 may be disposed onthe second surface 1122 of the second sheet 1120 opposite the pinningposts 1150. The pinning posts 1150 may provide features on the firstsurface 1111 with which a droplet 1160 may temporarily bind with andthereby limit the movement of the droplet 1160. The droplet 1160 mayinitially be disposed away from the heater 1140 and the pinning posts1150.

FIG. 11B, shows another view of the microfluidic device 1100. In FIG.11B, a compression force may be provided to the first sheet 1110 and/orthe second sheet 1120 to decrease the gap 1130 and move the droplet 1160toward the heater 1140 and the pinning posts 1150.

FIG. 11C shows another view of the microfluidic device 1100. In FIG.11C, the droplet 1160 is positioned in contact with the pinning posts1150 and the compression force removed. Thus, the first sheet 1110 andthe second sheet 1120 may return to an initial position and the droplet1160 is positioned over the heater 1140. As the pinning posts 1150engage with the droplet 1160, movement of the droplet 1160 may bereduced. Reduced motion may be particularly advantageous when thedroplet 1160 is undergoing a procedure, such as heating by the heater1140.

FIG. 12 is a flowchart showing an example operation 1200 formanipulating a microfluidic droplet in conjunction with pinning posts.The operation 1200 is described below with respect to microfluidicdevice 1100 of FIGS. 11A-11C, however, the operation 1200 may beperformed by any other suitable system or device.

The operation 1200 begins in block 1202 as a microfluidic droplet 1160is moved via mechanical manipulation to a region of the microfluidicdevice 1100 that includes pinning posts 1150. The mechanicalmanipulation may include the use of compression forces as described withrespect to FIGS. 1A-1C and 2 .

Next, in block 1204 the microfluidic droplet 1160 in the region of thepinning posts 1150 is heated. In some examples, the heat may be providedby the heater 1140. Next, in block 1206 the microfluidic droplet 1160may be moved, via mechanical manipulation, away from the region of themicrofluidic device that includes the pinning posts 1150.

In some examples, a well may be disposed within a microfluidic device tocontain and process a microfluidic droplet. One example is described inconjunction with FIGS. 13A-13E and 14 .

FIG. 13A shows a portion of a microfluidic device 1300. As shown in FIG.13A, the microfluidic device 1300 may include a first sheet 1310, asecond sheet 1320, and a gap 1330. The first sheet 1310, the secondsheet 1320, and the gap 1330 may be examples of the first sheet 110, thesecond sheet 120, and the gap 130 of FIG. 1A. In addition, the secondsheet 1320 may include an opening 1325 that couples the gap 1330 to aheater 1350. The heater 1350 may be formed in the shape of a well 1355.Thus, the well 1355 may be coupled to the gap 1330 through the opening1325. As shown, a droplet 1340 may be positioned away from the opening1325.

For ease of use, the second sheet 1320 may be disposed below (e.g.,closer to the ground) than the first sheet 1310. Such a configurationmay allow gravity to assist in receiving or moving the droplet 1340 inthe well 1355. In some other examples, an opening and well may bedisposed on the first sheet 1310. In such configurations, surfacetension and/or capillary action may cause the droplet 1340 to remain inthe well, despite the well 1355 being inverted.

FIG. 13B shows another view of the microfluidic device 1300. In FIG.13B, the droplet 1340 is moved, via mechanical manipulation, toward theopening 1325 in the second sheet 1320. For example, a compression forcemay be applied to the first sheet 1310 and/or the second sheet 1320 toreduce the gap 1330 and cause the droplet 1340 to move.

FIG. 13C shows another view of the microfluidic device 1300. In FIG.13C, the droplet 1340 within the well 1355 of the heater 1350. Thecompression force may be removed or reduced allowing the first sheet1310 and the second sheet 1320 to return to an initial position. Thewell 1355 may advantageously restrict movement of the droplet 1340,particularly while being heated by the heater 1350.

FIG. 13D shows another view of the microfluidic device 1300. In FIG.13D, the droplet 1340 is being drawn out of well 1355 of the heater 1350via mechanical manipulation. For example, a compression force may beapplied to the first sheet 1310 and/or the second sheet 1320 to reducethe gap 1330 and contact the droplet 1340. In some examples, thecompression force may be applied to a region of the microfluidic device1300 associated with a direction to receive the droplet 1340.

FIG. 13E shows the compression force removed or reduced from themicrofluidic device 1300. The first sheet 1310 and the second sheet 1320may return to an initial position and the droplet 1340 may be positionedaway from the well 1355 and the heater 1350.

FIG. 14 is a flowchart showing an example operation 1400 formanipulating a microfluidic droplet in conjunction with a well. Theoperation 1400 is described below with respect to the microfluidicdevice 1300 of FIGS. 13A-13E, however the operation 1400 may beperformed by any other suitable system or device.

The operation begins in block 1402 as a microfluidic droplet is moved,via mechanical actuation, into a well. For example, a compression forcemay be applied to the first sheet 1310 or the second sheet 1320 to causethe microfluidic droplet 1340 into the well 1355.

Next, in block 1404 the microfluidic droplet 1340 may be heated in thewell. For example, the heater 1350 may heat the microfluidic droplet1340 within the well 1355. Next, in block 1406, the microfluidic droplet1340 may be moved, via mechanical manipulation, away from the well. Forexample, a compression force may be applied to the first sheet 1310and/or the second sheet 1320 to reduce the gap 1330 and cause themicrofluidic droplet 1340 to come out of the well 1355.

FIG. 15A shows a portion of a microfluidic device 1500. As shown in FIG.15A, the microfluidic device 1500 may include a first sheet 1510, asecond sheet 1520, a gap 1530, and an electrode 1550. The first sheet1510, the second sheet 1520, and the gap 1530 may be examples of thefirst sheet 110, the second sheet 120, and the gap 130 of FIG. 1A. Theelectrode 1550 may be coupled to electrical circuits and the like (notshown) that provide high energy electric fields associated with causingelectroporation (the creation of temporary pores or openings within cellmembranes). As shown, a droplet 1540 may be positioned away from theelectrode 1550.

FIG. 15B shows another view of the microfluidic device 1500. In FIG.15B, a compression force may be applied to the first sheet 1510 and/orthe second sheet 1520. The compression force (e.g., mechanicalactuation) may reduce the gap 1530 and cause the droplet 1540 to movetoward the electrode 1550.

FIG. 15C shows another view of the microfluidic device 1500. In FIG.15C, the compression force is removed or reduced thereby allowing thefirst sheet 1510 and/or the second sheet 1520 to return to an initialposition. The mechanical actuation of FIG. 15B has positioned thedroplet 1540 on the electrode 1550. The droplet 1540 may undergoelectroporation using the electrode 1550.

FIG. 16 is a flowchart showing an example operation 1600 for providingelectroporation. The operation 1600 is described with respect to themicrofluidic device 1500 of FIGS. 15A-15C, however the operation 1600may be performed by any other suitable system or device.

The operation 1600 begins in block 1602 where a microfluidic droplet ismoved, via mechanical actuation, onto an electroporation electrode. Forexample, a compression force may be applied to the first sheet 1510and/or the second sheet 1520 to move the microfluidic droplet 1540 ontothe electrode 1550.

Next, in block 1604, the electrode 1550 provides an electric field tothe microfluidic droplet 1540. The electric field may be a high-powerelectric field provided by one or more circuits and devices. Theelectric field may temporarily provide openings or “pores” in the cellwalls of cellular material within the microfluidic droplet 1540.

Next, in block 1606, the microfluidic droplet is moved, via mechanicalmanipulation, away from the electrode. For example, compressive forcesmay be used in conjunction with the first sheet 1510 and the secondsheet 1520 to move the microfluidic droplet away from the electrode1550.

FIG. 17 shows an example microfluidic system 1700. The microfluidicsystem 1700 may include a cartridge 1710, a pressure actuator 1720, amagnet 1760, a heater 1770, and a controller 1780. In some examples, thepressure actuator 1720, the magnet 1760, the heater 1770, and thecontroller 1780 may be included with a housing or base station that maycouple to the cartridge 1710.

The cartridge 1710 may be an example of the microfluidic devices 100,300, 500, 700, 900, 1100, 1300, and 1500 of FIGS. 1, 3, 5, 7, 9, 11, 13,and 15 , respectively. The cartridge 1710 may include an input port1711, a first sheet 1712, a second sheet 1714, an optical sensor 1718,one or more pinning posts 1730, one or more electrodes 1740 and a firstheater 1750. The first sheet 1712 and the second sheet 1714 may behydrophobic and oleophobic sheets or may include a hydrophobic andoleophobic layer on one or more surfaces. The first heater 1750 may becoupled to a gap 1716 through an opening 1715 in and the second sheet1714. The first heater 1750 may form a well 1755. One or more dropletsmay be inserted into the cartridge 1710 through the input port 1711.Although only one input port 1711 is shown, in other examples thecartridge 1710 may include any feasible number of input ports.

The pressure actuator 1720 may contact or otherwise be coupled to thecartridge 1710. As shown, the pressure actuator 1720 may be coupled tothe first sheet 1712. In other examples, the pressure actuator 1720 maybe coupled to the second sheet 1714. The pressure actuator 1720 may alsobe coupled to the controller 1780. The controller 1780 may cause thepressure actuator 1720 to selectively apply one or more compressiveforces to the first sheet 1712 and/or the second sheet 1714. Thecompressive forces may manipulate the position of any droplets withinthe gap 1716. The optical sensor 1718 may detect the presence and/orlocation of a droplet (e.g., a microfluidic droplet) within the gap1716. The optical sensor 1718 may be coupled to the controller 1780. Inthis manner, data from the optical sensor may be used to guide or directthe pressure actuator 1720.

The magnet 1760 may be disposed adjacent to or on the cartridge 1710.Operation of the magnet 1760 may be controlled by the controller 1780.Similarly, a second heater 1770 may be disposed adjacent to, or on thecartridge 1710 and may also be controlled by the controller 1780.

The controller 1780 may control operations of the microfluidic system1700. Thus, the controller 1780 may control operations of the pressureactuator 1720, the magnet 1760, the first and second heaters 1750 and1770 and the one or more electrodes 1740 to perform one or moreoperations described herein.

FIG. 18A shows a portion of another microfluidic device 1800. Themicrofluidic device 1800 may include a first sheet 1810, a second sheet1820, and a stylus 1850. The first sheet 1810 may be separated from thesecond sheet 1820 by a gap 1830. The first sheet 1810, the second sheet1820 and the gap 1830 may be other examples of the first sheet 110, thesecond sheet 120 and the gap 130 of FIG. 1A. The stylus 1850 mayselectively provide a compression force to either the first sheet 1810or the second sheet 1820 (as shown, the stylus 1850 is selectivelyproviding a compression force to the first sheet 1810). The compressionforce from the stylus 1850 may selectively reduce the gap 1830 in someregions of the microfluidic device 1800 to move a droplet 1840 asdescribed above in conjunction with FIGS. 1-17 . For example, a positionand compression force of the stylus 1850 may be controlled by thecontroller 1780 of FIG. 17 .

In some examples, the stylus tip area may be selected to correspond toan anticipated size of a droplet 1840. The droplet 1840 may be anyfeasible droplet, including any feasible microfluidic droplet. Thus, thedroplet 1840 may be between 10⁻⁶ and 10⁻¹⁵ liters. Furthermore, in someexamples, the tip of the stylus may be shaped or treated to avoidscratching and/or abrading the surface of the first sheet 1810 or thesecond sheet 1820. For example, the tip of the stylus 1850 may include aroller to apply the compression force to the first sheet 1810. In someother examples, the tip of the stylus 1850 may be coated with alubricant.

FIG. 18B shows the stylus 1850 and possible associated end profiles1860. The end profiles 1860 are not meant to be limiting (e.g., the endprofiles 1860 are not an exhaustive listing of all possible endprofiles) but are instead meant to be exemplary. Thus, other endprofiles for the stylus 1850 are possible. Some end profiles 1860 maymore effectively move or manipulate the droplet 1840. For example,circular, rectangular, or oval profiles may more effectively move thedroplet 1840.

FIG. 19A shows a portion of another microfluidic device 1900. Themicrofluidic device 1900 may include a first sheet 1910, a second sheet1920, a gap 1930, and a stylus 1950 which may be examples of the firstsheet 1810, the second sheet 1820, the gap 1830, and the stylus 1850 ofFIG. 18A. The stylus 1950 may include an insulated and/or exposedelectrode 1955. In some examples, the electrode 1955 may be disposedtoward a tip of the stylus 1950 that may contact at least one of thefirst sheet 1910 or the second sheet 1920.

In some examples, the electrode 1955 may be provided a voltage (e.g., anelectric potential) that attracts the droplet 1940. When the stylus 1950is placed in contract with at least one of the sheets of themicrofluidic device 1900 (shown here as the first sheet 1910), theassociated sheet can perform as or be a hydrophobic and oleophobicdielectric separating the electrode 1955 from the droplet 1940.

The applied or provided voltage may be sufficient to affect or controlsurface tension of the droplet 1940. In this manner, the stylus 1950 mayattract and/or move the droplet 1940 in the gap 1930 without applying acompression force, but instead by providing a voltage to the electrode1955, and then moving the position of the stylus 1950 with respect tothe first and second sheets 1910 and 1920, respectively.

FIG. 19B shows another view of the microfluidic device 1900. As shown,the stylus 1950 may be moved planarly with respect to the first sheet1910 and the second sheet 1920. When the stylus 1950 is moved planarlywhile the electrode 1955 is energized with a sufficient voltage, thedroplet 1940 may move to follow the stylus 1950. Thus, any of thedroplet manipulations described with respect to FIGS. 1-17 may beperformed by energizing the electrode 1955 and moving the stylus 1950instead of using a compressive force.

FIG. 20 is a flowchart showing an example operation 2000 formanipulating a microfluidic droplet. The operation 2000 is describedwith respect to the microfluidic device 1900 of FIGS. 19A and 19B,however the operation 2000 may be performed by any other suitable systemor device. The operation 2000 begins in block 2002 as an electricpotential is provided to an electrode disposed or coupled to a stylus.For example, a voltage may be provided to the electrode 1955 that isdisposed on or near a tip of the stylus 1950. In addition, the stylus1950 may be in contact with at least one sheet (e.g., the first sheet1910 or the second sheet 1920) of the microfluidic device 1900. Theapplied or provided voltage may be sufficient to affect or controlsurface tension of the droplet 1940.

Next, in block 2004, the stylus is moved. For example, the stylus 1950may be moved relative to the first sheet 1910 and the second sheet 1920.Since a sufficient voltage is applied or provided to the electrode 1955(in block 2002), the droplet 1940 may move in response to motion of thestylus 1950.

Next, in block 2006, the voltage or potential is removed from theelectrode. For example, in block 2004 the stylus 1950 may be moved toposition the droplet 1940 into a predetermined treatment zone. Since themovement is complete, the voltage or potential may be removed from theelectrode 1955.

FIG. 21A shows a portion of another microfluidic device 2100. Themicrofluidic device 2100 may include a first sheet 2110, a second sheet2120, a gap 2130, a stylus 2150, and an electrode 2155 which may beexamples of the first sheet 1910, the second sheet 1920, the gap 1930,the stylus 1950, and the electrode 1955 of FIG. 19A.

In contrast to the microfluidic device 1900, the microfluidic device2100 may use a combination of a compression forces and applied voltagesto manipulate a droplet 2140. As shown, the stylus 2150 may bepositioned to one side of the droplet 2140. To move the droplet 2140,the stylus 2150 may provide a compression force to reduce the gap 2130while a voltage is provided to the electrode 2155. In this manner, thedroplet 2140 may be moved by a combination of compressive andelectromotive (e.g., voltage) forces. For example, while the stylus 2150is providing a compression force and a voltage is applied to theelectrode 2155, the stylus 2150 may be moved to change the position orlocation of the droplet 2140 within the gap 2130. The compression forcemay be provided to either the first sheet 2110 or the second sheet 2120.FIG. 21A shows one example as the stylus 2150 provides a compressionforce to the first sheet 2110.

FIG. 21B shows another view of the microfluidic device 2100. In thisview, the stylus 2150 has been moved to a new position with respect tothe first sheet 2110 and the second sheet 2120. The droplet 2140 hasbeen moved in response to previously provided compression and voltageprovided by and to the stylus 2150. Thus, in FIG. 21B the compressionforce is removed from the first sheet 2110 and the voltage is removedfrom the electrode 2155.

FIG. 22 is a flowchart showing an example operation 2200 formanipulating a microfluidic droplet. The operation 2200 is describedwith respect to the microfluidic device 2100 of FIGS. 21A and 21B,however the operation 2200 may be performed by any other suitable systemor device. The operation may begin in block 2202 as a compression forceis applied, via mechanical actuation, to at least one of the sheets of amicrofluidic device. For example, the stylus 2150 may provide acompression force to the first sheet 2110 and reduce the gap 2130 near adroplet 2140. The reduced gap may manipulate, at least in part, thedroplet 2140 between the first sheet 2110 and the second sheet 2120.

Next, in block 2204 an electric potential is provided to an electrodedisposed or coupled to the stylus. For example, a voltage may beprovided to the electrode 2155 that is disposed on or near the tip ofthe stylus 2150. The stylus 2150 may be in contact with at least onesheet of the microfluidic device 2100. The applied or provided voltagemay be sufficient to affect or control surface tension of the droplet2140.

Next, in block 2206, the stylus is moved. For example, the stylus 2150may be moved relative to the first sheet 2110 and the second sheet 2120.In this manner, the droplet 2140 may be manipulated or moved using acombination of compression forces provided by the stylus 2150 and avoltage applied to the electrode 2155.

Next, in block 2208 the electric potential is removed from theelectrode. For example, a voltage may be removed from the electrode2155. Then, in block 2210 the compression force is removed from at leastone sheet of the microfluidic device. For example, the stylus 2150 maybe moved away from the first sheet 2110 or the second sheet 2120.Because the droplet 2140 has been moved into a predetermined (e.g.,desired) position in block 2206, the compression force and voltage maybe removed from the stylus 2150.

In some examples, ferrous particles may be suspended within amicrofluidic droplet to assist with processing or assaying. After one ormore processing steps have been completed, the ferrous particles may beremoved from the droplet for further processing.

FIG. 23A shows a portion of another microfluidic device 2300. Themicrofluidic device 2300 may include a first sheet 2310, a second sheet2320, a gap 2330, and a stylus 2350 which may be examples of the firstsheet 1910, the second sheet 1920, the gap 1930, and the stylus 1950 ofFIG. 19A. In addition, the stylus 2350 may include a magnet 2356. Themagnet 2356 may be controllable. For example, the magnet 2356 may be anelectromagnet that may be enabled and disabled through a controlvoltage. In another example, the magnet 2356 may be movable. Thus, themagnet 2356 may be moved toward a tip of the stylus 2350 (as shown) oraway from the tip of the stylus 2350 (not shown). In this manner,magnetic field strength at or near the tip of the stylus 2350 may becontrolled and/or variable. A plurality of ferrous particles 2341 may bedistributed (suspended) throughout a droplet 2340.

FIG. 23B shows another view of the microfluidic device 2300. In thisview, the magnet 2356 is activated or enabled. For example, the magnet2356 may be an electromagnet that may be enabled through the applicationof power. In another example, the magnet 2356 may be a permanent magnetthat may be moved toward the droplet 2340. The magnet 2356 may attractor cause the ferrous particles 2341 to collect into one or more ferrousbeads 2342. Thus, the ferrous particles 2341 may come out of suspensionfrom the droplet 2340.

FIG. 23C shows another view of the microfluidic device 2300. While themagnet 2356 is activated or enabled, the stylus 2350 is moved. Themovement of the stylus 2350 may filter or remove the ferrous beads 2342from the droplet 2340.

FIG. 24 is a flowchart showing an example operation 2400 for removingsuspended ferrous particles from a microfluidic droplet. The operation2400 is described with respect to the microfluidic device 2300 of FIGS.23A-23C, however the operation 2400 may be performed by any othersuitable system or device.

The operation 2400 begins in block 2402 as a stylus with a magnet ismoved to a region proximate to a droplet with suspended ferrousparticles. For example, a stylus 2350 with a magnet 2356 may be movednear a droplet 2340 that includes suspended ferrous particles 2341.

Next, in block 2404 a magnet within or on the stylus is enabled. Forexample, the magnet 2356 may be an electromagnet that may be enabledthrough the application of power. In another example, the magnet 2356may be moved toward the tip of the stylus 2350. In this manner, themagnet 2356 may cause the ferrous particles 2341 to fall out ofsuspension and collect toward the magnet 2356. In some cases, thecollected ferrous particles 2341 may form a ferrous bead 2342.

Next, in block 2406, the stylus may be moved away from the droplet. Forexample, moving the stylus 2350 may move the ferrous bead 2342 out ofthe droplet 2340 thereby filtering the ferrous particles 2341 out of thedroplet 2340.

In some examples, ferrous material may be placed back into suspensionwithin a droplet through mechanical actuation. Another such example isdescribed below in conjunction with FIGS. 25-26 .

FIG. 25A shows a portion of another microfluidic device 2500. Themicrofluidic device 2500 may include a first sheet 2510, a second sheet2520, a stylus 2550, and a magnet 2556. The first sheet 2510, the secondsheet 2520, the stylus 2550, and the magnet 2556 may be examples of thefirst sheet 2310, the second sheet 2320, the gap 2330, the stylus 2350and the magnet 2356 of FIG. 23A. In some examples, the magnet 2556 maybe disengaged by either moving the magnet 2556 away from the tip of thestylus 2550 or by removing power if the magnet 2556 is an electromagnet.

A droplet 2540 may include non-dispersed ferrous or non-ferrousparticles. A compression force may be applied to the first sheet 2510and/or the second sheet 2520 (not shown) that can compress or deform thedroplet 2540. In some cases, a compression force may be applied to acenter or middle of the droplet 2540 by the stylus 2550.

FIG. 25B shows another view of the microfluidic device 2500. Thecompression force may be removed or reduced from the first sheet 2510and/or the second sheet 2520. Removal or release of the compressionforce may allow the droplet 2540 to return to a non-compressed ornon-deformed state. Transitioning from a compressed to a non-compressedstate (or vice-versa) may cause one or more ferrous or non-ferrousparticles 2541 within the droplet 2540 to become suspended. In somecases, the compression force may be applied and/or removed quickly orabruptly. Sudden application and/or removal of compression forces mayassist in dispersing ferrous and non-ferrous particles 2541 throughoutthe droplet 2540. In some cases, the compression force may be repeatedlyapplied and removed to disperse particles more evenly.

FIG. 26 is a flowchart showing an example operation 2600 for dispersingparticles in a microfluidic droplet. The operation 2600 is describedbelow with respect to the microfluidic device 2500 of FIGS. 25A and 25B,however the operation 2600 may be performed by any other suitable systemor device.

The operation 2600 begins in block 2602 where a magnet is disabled. Forexample, the magnet 2556 may be disabled either by moving the magnet2556 away from the tip of the stylus 2550 or by removing power from anelectromagnet comprising magnet 2556.

Next, in block 2604 a compression force is applied via mechanicalactuation, to a microfluidic droplet that includes ferrous and/ornon-ferrous particles that are to be suspended. For example, acompression force may be provided by a mechanical actuation force thatmay be applied by the stylus 2550 to the first sheet 2510 and/or thesecond sheet 2520 and also to the droplet 2540. The compression forcemay cause the droplet 2540 to deform or spread.

Next, in block 2606 the compression force may be released or reduced tosuspend the ferrous and/or non-ferrous particles in the droplet. Forexample, the removal or reduction of the compression force may cause themicrofluidic droplet 2540 to return to a spherical or quasi-sphericalshape that causes ferrous or non-ferrous particles to become at leastpartially become suspended within the droplet 2540.

In some cases, additional agitation of the microfluidic droplet may bedesired to enhance the distribution of the particles in the microfluidicdroplet 2540. To provide the additional agitation, a compression forcemay be repeatedly applied and removed (or reduced) for a predeterminednumber of cycles. Therefore, in block 2608, the number of completedcompression cycles is determined. A completed compression cycle mayinclude the application and removal or reduction of a compression force.If the number of compression cycles is less than a predetermined number,then the operation 2600 may return to block 2604. On the other hand, ifthe number of compression cycles is greater than or equal to thepredetermined number, then the operation 2600 may end.

Next, in block 2604 a compression force is applied via mechanicalactuation, to a microfluidic droplet that includes ferrous and/ornon-ferrous particles 2541 that are to be suspended. The compressionforce may be provided by a mechanical actuation force that may beapplied to the first sheet 2510 and/or the second sheet 2520 through thestylus 2550. The compression force may cause the droplet 2540 to deformor spread.

Next, in block 2604 the compression force may be released or reduced tosuspend the ferrous and/or non-ferrous particles 2541 in the droplet2540. The removal or reduction of the compression force may cause thedroplet 2540 to return to a spherical or quasi-spherical shape thatcauses ferrous or non-ferrous particles to become at least partiallybecome suspended within the droplet 2540.

In some examples, a stylus may include or be coupled to a reservoir orother liquid container that may be used to hold liquids aspirated from agap of a microfluidic device. Example devices are described below inconjunction with FIGS. 27-38 .

FIG. 27A shows a portion of another microfluidic device 2700. Themicrofluidic device 2700 may include a first sheet 2710, a second sheet2720, and a gap 2730. The first sheet 2710, the second sheet 2720, andthe gap 2730 may be examples of the first sheet 1910, the second sheet1920, and the gap 1930 of FIG. 19A. The first sheet 2710 may include apre-slit septa or other configurable opening (not shown) that may enablea droplet 2740 to be aspirated (removed) from the gap 2730.

FIG. 27B shows another view of the microfluidic device 2700. A septa2711 may be included on one of the sheets of the microfluidic device2700. As shown, the septa 2711 is located on (included with) the firstsheet 2710. In some examples, the septa 2711 may remain closed undermost operating conditions. For example, a compressive force may beapplied to the first sheet 2710 near the septa 2711. However, the septa2711 may remain substantially closed under the application of thecompressive force. In other words, the septa 2711 may have a closingforce to prevent liquid from “leaking” from the first sheet 2710. Thedroplet 2740 may be moved under the septa 2711.

FIG. 27C shows another view of the microfluidic device 2700. A stylus2750 may be positioned substantially over the septa 2711. The stylus2750 may include a lumen 2751 that may be coupled to the septa 2711 andconfigured to receive the droplet 2740. In some examples, a negativepressure may be provided to and/or through the stylus 2750 to aspiratethe droplet 2740 through the septa 2711 and into the stylus 2750. Thestylus 2750 may now move the droplet 2740 to other locations within themicrofluidic device 2700.

FIG. 28 is a flowchart showing an example operation 2800 for aspiratingliquids in a microfluidic device. The operation 2800 is described belowwith respect to the microfluidic device 2700 of FIGS. 27A-27C, howeverthe operation 2800 may be performed by any other suitable system ordevice.

The operation 2800 begins in block 2802 where a droplet is disposedadjacent to a septa. For example, the droplet 2740 may be moved to besubstantially under the septa 2711 though any feasible operationdisposed herein.

Next, in block 2804 a stylus is moved over the septa. For example, thestylus 2750 may be moved to be over the septa 2711. The stylus 2750 mayinclude a lumen 2751 that may couple to the septa 2711.

Next, in block 2806, a droplet is aspirated into the stylus. Forexample, a negative pressure may be applied by or through the stylus2750. The negative pressure may draw the droplet 2740 into the lumen2751.

In some examples, liquid may be provided or returned to a microfluidicdevice through a septa. Some examples are described below in conjunctionwith FIGS. 29 and 30 .

FIG. 29A shows a portion of another microfluidic device 2900. Themicrofluidic device 2900 may include a first sheet 2910, a second sheet2920, a gap 2930, and a stylus 2950. The first sheet 2910, the secondsheet 2920, the gap 2930, and the stylus 2950 may be examples of thefirst sheet 2710, the second sheet 2720, the gap 2730, and the stylus2750 of FIG. 27A. Thus, first sheet 2910 may include a septa 2911.

In some examples, liquid that may be included within the stylus 2950 maybe injected through the septa 2911 and into the gap 2930. Prior toinjecting the liquid, the stylus 2950 may be positioned over the septa2911. In some examples, a lumen 2951 of the stylus 2950 may be coupledto the septa 2911. A positive pressure may be applied to the lumen 2951to push the liquid out of the stylus 2950 and form a droplet 2940 withinthe gap 2930. Thus, the applied pressure may overcome a closing force ofthe septa 2911.

FIG. 29B shows another view of the microfluidic device 2900. The stylus2950 may be moved away from the droplet 2940. Furthermore, a compressiveforce may be provided by the stylus 2950 to the first sheet 2910 (and/orthe second sheet 2920) to reduce the gap 2930. The reduced gap 2930 maycause the droplet 2940 to move relative to the first sheet 2910 and thesecond sheet 2920.

FIG. 29C shows another view of the microfluidic device 2900. The stylus2950 may move to move the droplet 2940. After the motion of the droplet2940 is complete, then the stylus 2950 may be positioned to remove thecompression source to the first sheet 2910 and/or the second sheet 2920.Thus, the stylus 2950 may operate as a pipette to move liquids (e.g.,the droplet 2940) within the microfluidic device 2900.

FIG. 30 is a flowchart showing an example operation 3000 formanipulating liquids in a microfluidic device. The operation 3000 isdescribed below with respect to the microfluidic device 2900 of FIGS.29A-29C, however the operation 3000 may be performed by any othersuitable system or device.

The operation 3000 begins in block 3002 where a droplet is dispensedfrom a stylus through a septa and into a gap of a microfluidic device.For example, the stylus 2950 may be positioned over the septa 2911. Apositive pressure may be applied to the stylus 2950 and/or lumen 2951 toinject a liquid through the septa 2911 into the gap 2930.

Next, in block 3004 the distance between the two sheets is selectivelyreduced, via mechanical actuation, near the microfluidic droplet. Forexample, the reduced distance may result in a reduced gap 2930 betweenthe first sheet 2910 and the second sheet 2920. In some examples, acompression force may be provided by the stylus 2950 adjacent to (e.g.,next to) one side of the droplet 2940. The compression force may reducethe gap 2930 causing the droplet 2940 to move toward the compressionforce. In some variations, the compression force may deform one end ofthe droplet 2940.

Next, in block 3006 the distance between the two sheets is restored. Forexample, the compression force applied in block 3004 may be removedallowing the first sheet 2910 and/or the second sheet 2920 to return toits initial separation distance. In some examples, the distance betweenfirst sheet 2910 and second sheet 2920 is returned to a distance similarto the initial separation distance associated with block 3602.

In some examples, a stylus may include additional devices or systemsthat may be used to process a droplet within the stylus. Some examplesare described below in conjunction with FIGS. 31-38 .

FIG. 31A shows a portion of another microfluidic device 3100. Themicrofluidic device 3100 may include a first sheet 3110, a second sheet3120, a gap 3130, and a stylus 3150. The first sheet 3110, the secondsheet 3120, and the gap 3130 may be examples of the first sheet 1810,the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3150 mayinclude a temperature control element 3155. Through the temperaturecontrol element 3155, any liquid that is aspirated from the gap 3130 maybe heated within the stylus 3150 as part of a chemical assay or otherprotocol or process. For example, a droplet 3140 may be disposed beneatha septa 3111 that is included in the first sheet 3110. The stylus 3150may be disposed over the septa 3111. In some examples, the temperaturecontrol element 3155 may provide heating or cooling to surrounding areabased on a control voltage, signal, or the like.

FIG. 31B shows another view of the microfluidic device 3100. The droplet3140 may be aspirated from the gap 3130, through the septa 3111, andinto the stylus 3150. In this manner, while the droplet 3140 is withinthe stylus 3150, the droplet 3140 may undergo temperature processing asprovided by the temperature control element 3155. In some examples, atemperature sensor (not shown) may be included with the stylus 3150and/or the temperature control element 3155. A controller (not shown)may monitor and control the temperature of the droplet 3140 as part ofthe temperature processing. For example, the controller may monitor thetemperature of the droplet 3140 and control the temperature provided bythe temperature control element 3155.

FIG. 31C shows another view of the microfluidic device 3100. Aftertemperature processing, the droplet 3140 may be injected into the gap3130 through the septa 3111 in the first sheet 3110.

FIG. 32 is a flowchart showing an example operation 3200 for thermallytreating liquids in a microfluidic device. The operation 3200 isdescribed below with respect to the microfluidic device 3100 of FIGS.31A-31C, however the operation 3200 may be performed by any othersuitable system or device.

The operation begins in block 3202 where a stylus is positioned over asepta and a droplet. For example, the stylus 3150 may be positioned overthe septa 3111 which is over a droplet 3140.

Next, in block 3204, the droplet is aspirated into the stylus throughthe septa. For example, a negative pressure may be provided to thestylus 3150. In response, the droplet 3140 may be drawn through thesepta 3111 and into the stylus 3150.

Next, in block 3206, the temperature of the droplet is controlled. Forexample, a temperature control element 3155 may be used to monitor andcontrol the temperature of the droplet 3140 which is contained withinthe stylus 3150.

Next, in block 3208, the droplet is injected into the gap through thesepta. For example, temperature processing may be complete. A positivepressure may be provided to the stylus 3150 causing the droplet 3140 tobe injected though the septa 3111 and into the gap 3130. In someexamples, the stylus 3150 may be repositioned with respect to the firstsheet 3110 and/or the second sheet 3120 prior to returning the droplet3140 to the gap 3130.

FIG. 33 shows a portion of another microfluidic device 3300. Themicrofluidic device 3300 may include a first sheet 3310, a second sheet3320, a gap 3330, and a stylus 3350. The first sheet 3310, the secondsheet 3320, and the gap 3330 may be examples of the first sheet 1810,the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3350 mayinclude a temperature control element 3355. The temperature controlelement 3355 may be disposed toward a portion of the stylus 3350 thatmay contact the first sheet 3310. The stylus 3350 and the temperaturecontrol element 3355 are positioned on the first sheet 3310 over adroplet 3340. In this manner, the temperature control element 3355, mayprovide heat for any temperature controlled processing to the droplet3340 through the first sheet 3310.

In some examples, the temperature control element 3355 may include atemperature sensor (not shown). In this manner a controller (also notshown) may control the temperature of the droplet 3340 to be within adesired temperature.

FIG. 34A shows a portion of another microfluidic device 3400. Themicrofluidic device 3400 may include a first sheet 3410, a second sheet3420, a gap 3430, and a stylus 3450. The first sheet 3310, the secondsheet 3320, and the gap 3330 may be examples of the first sheet 1810,the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3450 mayinclude a light sensor 3455 and a light source 3456. In some examples, areaction that may have occurred or be occurring within a droplet 3440may be monitored by sensing light that may be transmitted or reflectedthrough the droplet 3440. Thus, the droplet 3440 may be drawn into thestylus 3450 where transmitted and/or reflected light can be detectedand/or measured. The amount of detected light may be associated with theprogress or completion of a reaction.

In some examples, to measure a reaction, the stylus 3450 may bepositioned over a septa 3411 that is included in the first sheet 3410.Additionally, the droplet 3440 may be located under the septa 3411.

FIG. 34B shows another view of the microfluidic device 3400. As shown,the droplet 3440 may be aspirated through the septa 3411 and into thestylus 3450. The light source 3456 may emit light into the droplet 3440.The light source 3456 may be any feasible solid state and/orincandescent light source. The light may be reflected and/or transmittedthrough the droplet 3440. This light may be detected by the light sensor3455. The light sensor 3455 may be any feasible light sensor or detectorsuch as a photo diode or the like. The amount of detected light may beassociated with the progress or completion of a reaction. In thismanner, the detected light may indicate the progress or completion of areaction.

FIG. 34C shows another view of the microfluidic device 3400. As shown,the droplet 3440 may be returned to the gap 3430. In some examples, thestylus 3450 may inject the droplet 3440 through the septa 3411 in thefirst sheet 3410. In this manner, after determining the lighttransmission or refraction of the droplet 3440, the droplet 3440 may bereturned to the gap 3430 for other processing.

FIG. 35 is a flowchart showing an example operation 3500 for performinga reaction measurement. The operation 3500 is described below withrespect to the microfluidic device 3400 of FIGS. 34A-34C, however theoperation 3500 may be performed by any other suitable system or device.

The operation begins in block 3502 where a stylus is positioned over asepta and a droplet. For example, the stylus 3450 may be positioned overthe septa 3411 which is over a droplet 3440.

Next, in block 3504, the droplet is aspirated into the stylus throughthe septa. For example, a negative pressure may be provided to thestylus 3450. In response, the droplet 3440 may be drawn through thesepta 3411 and into the stylus 3450.

Next, in block 3506, the reaction of the droplet is monitored. Forexample, a light source 3456 may be used to emit light in the stylus3450 and into the droplet 3440. A light sensor 3455 may detecttransmitted and/or reflected light from the droplet 3440. In some cases,the transmitted and/or reflected light may be associated with an amountof progress of a reaction occurring within the droplet 3440.

Next, in block 3508, the droplet is injected into the gap through thesepta. For example, reaction monitoring may be complete. A positivepressure may be provided to the stylus 3450 causing the droplet 3440 tobe injected though the septa 3411 and into the gap 3430. In someexamples, the stylus 3450 may be repositioned with respect to the firstsheet 3410 and/or the second sheet 3420 prior to returning the droplet3440 to the gap 3430.

FIG. 36 shows a portion of another microfluidic device 3600. Themicrofluidic device 3600 may include a first sheet 3610, a second sheet3620, a gap 3630, and a stylus 3650. The first sheet 3610, the secondsheet 3620, and the gap 3630 may be examples of the first sheet 1810,the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3650 mayinclude a light source 3655. The light source 3655 may be disposedtoward a portion of the stylus 3650 that may contact the first sheet3610.

Opposite the light source 3655 may be a light detector 3621. As shown,the light detector 3621 may be disposed on the second sheet 3620. When adroplet 3640 is disposed between the light source 3655 and the lightdetector 3621, the light detector 3621 may detect transmitted and/orreflected light. Thus, the light source 3655 and the light detector 3621may monitor progress of a reaction, similar to as described with respectto FIGS. 34 and 35 . In some examples the position of the light source3655 and the light detector 3621 may be reversed. In other words, thelight sensor 3621 may be included with the stylus 3650 and the lightsource 3655 may be disposed on the second sheet 3620.

FIG. 37A shows a portion of another microfluidic device 3700. Themicrofluidic device 3700 may include a first sheet 3710, a second sheet3720, a gap 3730, and a stylus 3750. The first sheet 3710, the secondsheet 3720, and the gap 3730 may be examples of the first sheet 1810,the second sheet 1820, and the gap 1830 of FIG. 18A. The stylus 3750 mayinclude a sonication probe 3755. In some examples, the sonication probe3755 may deliver sonic and/or ultrasonic stimulation to a droplet 3740.The sonication probe 3755 may be any feasible piezoelectric device.

In some examples, to deliver sonic or ultrasonic stimulation, the stylus3750 may be positioned over a septa 3711 that is included in the firstsheet 3710. Additionally, the droplet 3740 may be located under thesepta 3711.

FIG. 37B shows another view of the microfluidic device 3700. As shown,the droplet 3740 may be aspirated through the septa 3711 and into thestylus 3750. Once the droplet 3740 is in the stylus 3750, the sonicationprobe 3755 may be activated or enabled allowing sonic and/or ultrasonicwaves to be provided to the droplet 3740. The delivery sonic stimulationmay be controlled by a controller (not shown).

FIG. 37C shows another view of the microfluidic device 3700. As shown,the droplet 3740 may be returned to the gap 3730. In some examples, thestylus 3750 may inject the droplet 3740 through the septa 3711 in thefirst sheet 3710. In this manner, after delivering sonic stimulation tothe droplet 3740, the droplet 3740 may be returned to the gap 3730 forother processing.

FIG. 38 is a flowchart showing an example operation 3800 for performinga sonic treatment. The operation 3800 is described below with respect tothe microfluidic device 3700 of FIGS. 37A-37C, however the operation3800 may be performed by any other suitable system or device.

The operation begins in block 3802 where a stylus is positioned over asepta and a droplet. For example, the stylus 3750 may be positioned overthe septa 3711 which is over a droplet 3740.

Next, in block 3804, the droplet is aspirated into the stylus throughthe septa. For example, a negative pressure may be provided to thestylus 3750. In response, the droplet 3740 may be drawn through thesepta 3711 and into the stylus 3750.

Next, in block 3806, the sonic treatment may be delivered to thedroplet. For example, the sonication probe 3755 may deliver sonic and/orultrasonic treatment to the droplet 3740.

Next, in block 3808, the droplet is injected into the gap through thesepta. For example, sonic treatment may be complete. A positive pressuremay be provided to the stylus 3750 causing the droplet 3740 to beinjected though the septa 3711 and into the gap 3730. In some examples,the stylus 3750 may be repositioned with respect to the first sheet 3710and/or the second sheet 3720 prior to returning the droplet 3740 to thegap 3730.

FIG. 39 shows a block diagram of a device 3900 that may be one exampleof the any microfluidic device or system described herein. The device3900 may include a pressure actuator 3920, one or more magnets 3922, oneor more heaters 3924, one or more electrodes 3926, an optical sensor3927, a light source and sensor 3928, a sonic device 3929, a processor3930, and a memory 3940.

In some examples, the pressure actuator 3920, which is coupled to theprocessor 3930, may be used to provide forces, including compression andactuation forces to one or more sheets of a microfluidic cartridge. Insome examples, the pressure actuator 3920 may use mechanical, pneumatic,and/or electrical actuators to provide the compression and/or actuationforces. The compression and/or actuation forces may be provided througha controllable stylus. In some other examples, the pressure actuator3920 may provide positive and/or negative pressure to a lumen of astylus. In this manner a droplet may be aspirated from a gap of amicrofluidic device and drawn into the stylus.

The one or more magnets 3922, which are also coupled to the processor3930, may be used to selectively provide magnetic fields that may beused for and during microfluidic droplet manipulation. In some examplesthe magnet may be disposed adjacent to a side of a hydrophobic andoleophobic sheet. In some other examples, the magnet may be disposedwithin a stylus.

The one or more heaters 3924, which are also coupled to the processor3930, may be used to provide heat to one or more microfluidic droplets.The heat may be used during analysis or assay of the microfluidicdroplets. In some examples the heater (e.g., heating element) may bedisposed adjacent to a side of a hydrophobic and oleophobic sheet. Insome other examples, the heater may be disposed within a stylus.

The one or more electrodes 3926, which are also coupled to the processor3930, may be used to provide electric fields used for electroporation.In some examples, the processor 3930 may include one or more electricalcircuits or devices to generate large magnitude electric fields for theone or more electrodes 3926.

The optical sensor 3927, which is also coupled to the processor 3930,may detect the presence and/or position of any droplet, such as anymicrofluidic droplet disposed between two or more hydrophobic andoleophobic sheets.

The light source and sensor 3928, which are also coupled to theprocessor 3930, may provide light and detect a transmitted or reflectedlight associated with a microfluidic droplet. In some examples, thelight source and sensor 3928 may be included with a stylus. In someother examples, the light source and sensor 3928 may be disposed on aside of a hydrophobic and oleophobic sheet and the stylus.

The sonic device 3929, which is also coupled to the processor 3930, mayprovide sonic or ultrasonic treatment to a microfluidic droplet. In someexamples, the sonic device 3929 may be included with the stylus. In someexamples, the sone device 3929 may be a piezo electric device.

The processor 3930, which is also coupled to the memory 3940, may be anyone or more suitable processors capable of executing scripts orinstructions of one or more software programs stored in the device 3900(such as within the memory 3940).

The memory 3940 may include septa location data 3941. In some examples,the septa location data 3941 may be a database of the locations of oneor more septa openings that may be disposed on a hydrophobic andoleophobic sheet. Thus, the processor 3930 may use the septa locationdata 3941 to position the stylus over any particular septa.

The memory 3940 may also include a non-transitory computer-readablestorage medium (e.g., one or more nonvolatile memory elements, such asEPROM, EEPROM, Flash memory, a hard drive, etc.) that may store thefollowing software modules: a pressure actuator control module 3942 tocontrol the pressure actuator 3920; a magnet control module 3944 tocontrol the one or more magnets 3922; a heater control module 3946 tocontrol the one or heaters 3924; an electrode control module 3948 tocontrol the one or more electrodes 3926; an optical sensor controlmodule 3949 to control the optical sensor 3927; a sonic control module3950 to control sonic device 3929; and a light source and sense controlmodule 3951.

Each software module includes program instructions that, when executedby the processor 3930, may cause the device 3900 to perform thecorresponding function(s). Thus, the non-transitory computer-readablestorage medium of memory 3940 may include instructions for performingall or a portion of the operations described herein.

The processor 3930 may execute the pressure actuator control module 3942to manipulate one or more microfluidic droplets disposed between atleast two hydrophobic and oleophobic sheets by applying forces throughthe pressure actuator 3920. For example, execution of the pressureactuator control module 3942 may cause compressive, pinning, and/oractuation forces to be applied to at least one of the hydrophobic andoleophobic sheets. The forces may be selectively applied to move,separate, combine, and/or mix one or more microfluidic droplets. In someexamples, execution of the pressure control module 3942 may cause astylus to move across at least one of the hydrophobic and oleophobicsheets, apply a compression force, and cause a droplet to move.

The processor 3930 may execute the magnet control module 3944 toselectively control, enable, and/or disable one or more magnets 3922. Insome examples, execution of the magnet control module 3944 may causepower to be applied to electromagnets included in the magnets 3922. Insome examples, execution of the magnet control module 3944 may cause oneor more magnets 3922 to be moved closer to, or away from, one or moremicrofluidic droplets.

The processor 3930 may execute the electrode control module 3948 toselectively provide electric fields to the one or more electrodes. Forexample, execution of the electrode control module 3948 may provide oneor more large magnitude electric fields to the electrodes 3926 and causeelectroporation to occur on cell membranes within the microfluidicdroplet.

The processor 3930 may execute the optical sensor control module 3949 tocontrol and processes data from the optical sensor 3927. For example,execution of the optical sensor control module 3949 may cause theoptical sensor 3927 to receive or capture image data as well as causingthe processor 3930 to process the image data and determine the presenceand/or location of any droplets.

In some examples, the processor 3930 may use image data from the opticalsensor 3927 to control actions of the pressure actuator 3920. Forexample, the processor 3930 may process the image data with the pressureactuator control module 3942 and thereby guide the application ofcompressive forces on one or more hydrophobic and oleophobic sheets.

The processor 3930 may execute the sonic control module 3950 to controlthe sonic device 3929. For example, execution of the sonic controlmodule 3950 may activate or enable the sonic device 3929 therebyallowing or enabling the sonic device 3929 to provide sonic and/orultrasonic stimulation or treatment to a droplet.

The processor 3930 may execute the light source and sense control module3951 to control the light source and sensor 3928. For example, executionof the light source and sense control module 3951 may cause light to beemitted by a light source and transmitted or reflected light to besensed by a light detector. In this manner, a reaction or process may bedetected in accordance with the detected light.

Cartridges

As mentioned above, the cartridges described herein may generallyinclude a first (e.g., upper) sheet that is elastically deformable, asecond (e.g., lower) sheet, and a frame separating the two to form anair gap. The first sheet may be an elastomeric material, such as apolyester (e.g., TPE) natural rubber, synthetic rubber, nitrile rubber,silicone rubber, urethane rubbers, chloroprene rubber, Ethylene VinylAcetate (EVA), etc. The second sheet may be the same of a differentmaterial. The sheets of the cartridge are generally planar structuresand may be a membrane, a layer, etc. The first and second sheets may beheld in tension over or against the frame. In some examples, multipleframes may be used. The frame may be formed of any appropriate material,such as a rigid semi-rigid polyester.

FIGS. 40A-40D illustrate examples of a cartridge 4006, 4006′ asdescribed herein. The cartridge includes a frame 4017, a first sheet4007 and a second sheet 4009. In the example shown in FIGS. 40A, 40C and40D, the cartridge is divided into three lanes 4005, 4005′, 4005″. Inthis example the frame is configured as a divider with the threeseparate lanes. The example cartridge shown in FIG. 40B has eight lanes4015. Any appropriate number of lanes may be used (e.g., 1 lane, 2lanes, 3 lanes, 4 lanes, 5 lanes, 6 lanes, 7 lanes, 8 lanes, 9 lanes, 10lanes, 12 lanes, 15 lanes, 16 lanes, or more). The sectional view of acartridge shown in FIGS. 40C-40D illustrates an example with three lanesformed by the frame/divider 4017. The first sheet 4007 is held intension to the top of the frame, e.g., by welding and/or an adhesive4013. The second sheet 4009 in this example is also held in tension onthe frame and is welded and/or adhesively attached thereto. An air gap4011 is formed between the first sheet and the second sheet. In FIGS.40C and 40D, the portion of the cartridge is shown attached to a seatingportion of a mechanical microfluidics actuator having a recessed regionand a plurality of vacuum ports for sealing the second (bottom) sheetinto the seating region to make a tight thermal connection between thesecond sheet and the seating region. Sealing the second sheet 4009 tothe seat also expands the air gap 4011 to a larger height as compared tothe unattached configuration of the cartridge, as shown in FIG. 40D.

In the example shown in FIGS. 40A-40E, the initial height of the air gapmay be between about 0.5 mm and about 5 mm. In general, the height maybe between 0.1 mm and about 7 mm, and may be adjusted down or, as inFIGS. 40C and 40D, up. For example, compressing the first and/or secondsheet to form a 0.5 mm air gap height from a neutral height of about 3mm has been found to be very effective for mobility of droplets. In someexamples the neutral height of the air gap may be about 3 mm in height(spacing between the first and second sheets) which has been found to beeffective to move droplets without significant damage to the filmstested, as compared to larger gap spacing. At a heigh of about 3 mm, alarger droplet (e.g., 140 uL aqueous + 80 uL drop gloss) can touch thetop film with a small amount of compression. However, the height of thegap may be adjusted based on the volume of the droplet, and thematerials forming the first and second sheets.

FIG. 41 shows an exploded view of one examples of cartridge 4100. Inthis example, the cartridge includes a first (e.g., upper) frame 4117onto which the first sheet 4107 is attached in tension, so that it ispulled flat. All four sides of the sheet may be held in tension. Thesheet may be pinned, clamped, welded, adhesively attached, tacked, orotherwise secured to the frame. In this example, the first sheet 4107also includes a pair of openings 4133, 4133′, configured as input/outputwindows (“windows”) into which a fluid material (droplet, drop gloss,etc.) can be applied by manual or automatic means (e.g., pipetting,etc.). Fluid material may be added to the air gap through the windowadjacent to the sides of the window so that the mechanical microfluidicsactuator may use the force applicator adjacent to the window to pull thedroplet further into the air gap and manipulate the droplet (ordroplets) within the cartridge. The window 4133, 4133′ may be anyappropriate size. In some example the entire distal and/or proximal endof the cartridge may be open as a window (e.g., the first sheet mayextend just between two opposite sides of the frame (e.g., the firstframe). The edges of the window may be reinforced and/or smoothed. Insome examples the edges may be thickened (e.g., doubled over itself).The window may be any size, or ratio of the size of the surface of thefirst sheet. For example, the window may be between 50% and 100% of thewidth of the surface of the first sheet, and between about 0.1% to 10%of the length of the surface of the first sheet. In some examples thewindow is between 1 mm and 10 cm long and between 1 mm and 5 cm wide.Larger windows may be used. As mentioned above, in some examples thecartridge may also or alternatively include one or more smaller openingsfor applying/removing fluid (e.g., by pipetting).

The cartridge shown in FIG. 41 also includes a second frame 4121 towhich a second sheet 4109 is attached. The second sheet may be undertension or, as it may be configured to be secured to the base of themechanical microfluidics actuator, may be more loosely attached. Thesecond sheet 4109 may be attached to the second frame 4121 in anyappropriate manner. In this example, the first and second frames may bedisposable and the first and second sheets may be, e.g., TPE film, FEPfilm, etc. The first sheet may be adhesively attached to the first frameby, e.g., an adhesive such as a double-sided adhesive film (e.g., 3M300L SE, 2 mil thick double adhesive).

In FIG. 41 , the cartridge also includes a spacer frame 4119 that issandwiched between the first and second frames and the first and secondsheets. The first and second sheets may be attached or unattached to thespacer frame. The first frame and the second frame may be secured to thespacer frame. IN some examples the spacer frame is formed of ahydrophobic and oleophobic material, e.g., PTFE.

Alternatively, in some examples only a single frame, which may or maynot include spacers, may be used, and the first sheet may be attached tothe first side of the frame while the second sheet is attached to thesecond side of the frame. The frame of the cartridge shown in thisexample may be rigid; in some examples the frame may be flexible and/orhinged.

FIGS. 42A-42B illustrate another example of a portion of a cartridge. Inthis example, the cartridge is shown having a first frame 4217 to whichthe first sheet 4207 is attached. The first sheet may be attached to thetop or bottom of the first frame. In FIGS. 41 and 42A the sheet is shownattached to the bottom of the first frame. Frames may be formed in anyappropriate matter, including laser cutting, injection molding, etc.,and may be any appropriate material (e.g., polymeric material, such aspolyester, ABS or POM (glass filled)). FIG. 42B shows an enlarged sidesectional view of the first frame 4217 and first sheet 4207. In thisexample, the first sheet is a TPE film and is adhesively held to thefirst frame under tension using an adhesive 4210 (e.g., 3m 300LSE, 2 milthick, double-sided adhesive). In FIGS. 40 and 41A-41B, the first andsecond sheets are between about 20-60 µm thick (e.g., between 25-50 µmthick, etc.) and may be formed of an elastomeric material.

Any of these cassettes may include a frame (backbone) that ishydrophobic, e.g., polypropylene, and may include one or more internalstructures including, but not limited to spacer frames. For example, theapparatus may include posts (pinning posts), and/or an absorber(absorber material). The absorber may be used to remove waste (e.g.,from rinsing/washing, drop gloss, etc.). In some cases an edge of theframe may include an absorber. The frames may include markings,including computer readable markings (e.g., QR codes, bar codes, etc.)that may uniquely identify the cassette. The cassettes may be oriented,e.g., to allow positioning in the mechanical microfluidics actuator seatin a preferred or exclusive orientation, or they may be non-oriented(allowing application in any orientation). In some cases the cartridgemay include a specific “top” and “bottom” and may be marked or coded(including color coded) and or keyed to fit into the mechanicalmicrofluidics actuator seat with the upper surface “up”.

FIGS. 43A-43C illustrate the insertion of an example of a cartridge 4302(see FIG. 43A) into a mechanical microfluidics actuator seat (as shownin FIGS. 43B-43C). In this example the cartridge includes an elasticallydeformable upper sheet 4307 and an elastically deformable lower sheet4309, 4309′ that are both attached to a frame 4304 (e.g., molded polymerframe) to form an air gap 4312 having an initial air gap height 4319.The upper sheet and lower sheet in this example are attached to the sameframe 4304 and are shown to be adhesively attached 4314.

In FIG. 43B the cartridge is shown seated on the seat of the mechanicalmicrofluidics actuator 4322. The mechanical microfluidics actuator inthis example includes a recessed seating region 4320 that includemultiple vacuum ports coupled to a vacuum manifold 4322 coupled to asource of negative pressure and controlled by the controller of themechanical microfluidics actuator. In FIG. 43B the suction is shownapplied, pulling the lower sheet 4309 of the cartridge into continuouscontact with the seating region, and increasing the height of the airgap 4312 to a larger height 4319′ (as compared to the neutral height4319).

FIG. 43C illustrates the cartridge within the mechanical microfluidicsactuator shown in FIG. 43B with a droplet 4331 shown in the air gap4312. In this example, the upper and lower sheets 4307, 4309 may be,e.g., an elastomeric polyester and the cartridge frame 4304 may be amolded polyester.

FIGS. 44A-44B schematically show two alternative examples of cartridgesseated and secured to mechanical microfluidics actuator seating regions.In FIG. 44A the cartridge includes a frame 4406 to which a first sheet4407 and a second sheet 4409 are attached, separated from each other toform an air gap 4421. In FIG. 44A, similar to FIGS. 43B-43C, themechanical microfluidics actuator seating region 4419 includes vacuumports securing the lower (second) sheet 4409 against the surface of theseating region so that there is no gap between the seating region andthe second sheet, which is held immobilized against the seating region,as shown. In FIG. 44A the droplet 4412 may be heated/cooled or otherwisemanipulated through the bottom (second) sheet by applying thermal energythrough the particular sub-region of the seating region of themechanical microfluidics actuator. In this example the entire seatingregion is shown as recessed; in other examples only a portion of theseating region is recessed, forming a well (described below) into whichthe droplet may be held.

FIG. 44B shows another example of a cartridge held in a seating regionof a mechanical microfluidics actuator. In this example the cartridgeincludes a frame 4406 and an upper sheet 4407 and a lower sheet 4409;this cartridge is secured to the seating region by a securement such asa clamp 4420 applying a securing force against the frame to hold thecartridge immobile in place. In this example the mechanicalmicrofluidics actuator does not need to include a vacuum (e.g., vacuumports) to secure contact between the second sheet 4409 and the outerseating surface of the mechanical microfluidics actuator; in FIG. 44Bthe outer seating surface may be raised along the length of the air gap4421. Thus, any of the mechanical microfluidics actuators describedherein may not include a vacuum. Any of the mechanical microfluidicsactuator described herein may include a securement (e.g., clamp, lock,etc.) holding the cartridge against the mechanical microfluidicsactuator seating region.

As described in greater detail below, any of these mechanicalmicrofluidics actuators may include a controller, and one or morethermal regions that may locally heat/cool the seating region andtherefore a droplet within the air gap of the cartridge over thisportion of the seating region.

In general, any of the seating regions of the mechanical microfluidicsactuators may include a shape to which the lower sheet of the cartridgemay conform, which may have numerous benefits, such as securing thedroplet (e.g., pinning the droplet) to a particular region and/orenhancing the thermal energy transfer. FIGS. 44C-44E schematicallyillustrate examples in which the mechanical microfluidics actuatorseating region include a projection. In FIG. 44C the cartridge is shownseated on a mechanical microfluidics actuator 4419″ seating regionincluding a rail 4411 (e.g., rail region). The cartridge may be helddown by a securement (not shown, such as a clamp, magnet, etc.). Thedroplet 4412 between the first sheet 4407 and the second sheet 4409 istherefore held (e.g., by capillary force) within the center of the airgap 4421 region, and may be moved within the channel (e.g., in/out ofthe plane of the section shown) by a force applicator applying force toreduce the height of the air gap. FIG. 44D shows an example in which themechanical microfluidics actuator 4419‴ includes a step-up raid 4411′that deforms the lower sheet 4409′ slightly into the air gap 4421 buthas a wider base than the rail of FIG. 44C. In FIG. 44E the mechanicalmicrofluidics actuator 4419⁗ is similar to that shown in FIG. 44D butincludes a dome-shaped rail 4411″. Any of these examples may, but doesnot need to, including a vacuum manifold with vacuum ports to secure thesecond sheet 4409, 4409′, 4409″ to the outer surface of the seatingregion. Alternatively or additionally these apparatuses may include asecurement (such as a clamp, lock, magnetic securement, etc.) to holdthe cartridge in position.

In general, the methods and apparatuses described herein may include theuse of a rail region within the air gap. The rail region may generallyhave a gap width that is less than the gap width of a region of the airgap surrounding the rail region, e.g., around the periphery of the airgap, on either side of the rail region. The rail region may form anelevated bed. As mentioned above, the rail region may be formed bydeflecting the second (e.g., lower) sheet of the air gap; in someexamples the second sheet is more rigid than the first sheet (e.g., isformed of a relatively stiff material) and the rail region may be formedof the more rigid second sheet. Surprisingly, the droplet may avoid theregions in the periphery (adjacent to the rail region) having a greatergap width, which may prevent loss of the droplet volume, particularlywith smaller volume droplets (e.g., less than 15 µL, 10 µL or less, 5 µLor less, 1 µL or less, 1 µL or less, etc.).

Mechanical Microfluidics Actuator

As mentioned, any of these apparatuses may include a mechanicalmicrofluidics actuator. FIGS. 45A-45E illustrate one example of aportion of a mechanical microfluidics actuator. In FIG. 45A themechanical microfluidics actuator 4500 includes a seating region (seat)4531 onto which a cartridge may be secured. In the example shown, theseating region includes a plurality of parallel lanes 4532 (eight areshown) running the length of the seating region. The seat in thisexample includes a plurality of vacuum ports coupled to a vacuummanifold 4538 to apply a suction to conformably secure the lower(second) sheet of a cartridge to the seating region. In addition, eachof the lanes of the seating region includes a plurality of differentzones for thermal control 4533, magnetic field application 4435, or bothmagnetic field and thermal control 4542. The thermal control regions maybe in thermal communication with a heater/cooler (e.g., Peltier device),and the magnetic control regions may each include a local electromagnet.This is illustrated in FIG. 45B, in which the seating region 4531 hasbeen made transparent to show the thermal control zones 4533,electromagnet zones 4539 and combined thermal control/magnetic zones4542. In this example the base of the mechanical microfluidics actuatormay be a heat sink 4536 to allow local application of heating/cooling.FIG. 45C shows an examples of a seating region of a mechanicalmicrofluidics actuator with a cartridge 4506 attached to the seatingregion 4531.

Although this example includes three kinds of zones arranged in analternating pattern along the length of each lane of the seating region(which may correspond to lanes in the cartridge 4506, as shown in FIG.45C, other patterns of zones and/or other types of zones (e.g.,heating/cooling, magnetic, electrical energy, sensing/imaging, UVapplying, sonication application, etc.) may be included. FIGS. 45D and45E show examples of the seating region topology in slightly greaterdetail. For example, in FIGS. 45D and 45E the seating regions mayinclude a plurality of wells 4541 formed therein which may underlie athermal control region. For example, in FIG. 54D the mechanicalmicrofluidics actuator seating region including a plurality of thermalcontrol regions configured as wells 4541 having a shallow, bowl-shapeddepression which is formed of a thermally conductive material 4533. Thebowl also includes a suction port 4555 in communication with a suctionmanifold 4538 to hold down the second sheet 4509. The method of drivingthe droplet (e.g., with the force applicator) into the well may pindroplet in the well and may greatly reduce or limit evaporation,particularly when heating the droplet (e.g., for thermocycling thedroplet).

FIG. 46 schematically illustrates an example of a portion of anapparatus (such as the cartridge and mechanical microfluidics actuator)similar to that shown in FIGS. 45A-45E, including a vacuum port 4638securing the second sheet 4609 of the cartridge to the seating region ofthe mechanical microfluidics actuator 4631. In FIG. 46 a droplet 4612 isshown in the air gap region and is coated with a drop gloss 4652material. The drop gloss coating may be formed of a material that limitsevaporation and is immiscible with the droplet.

In general, any appropriately sized droplet may be used, includingmicroliter and sub-microliter droplets. However, in some cases it may bedifficult for smaller (e.g., less than 2 µL) to be transferred reliably.It may also be beneficial to use fluid transfer of droplets of any sizewithout requiring negative pressure (e.g., suction), e.g., withoutpipetting. FIG. 47 illustrates one example of a method for reliablytransferring a very small droplet, including (but not limited to)transferring into a cartridge as described herein. In this example asolid transfer member 4671 having a concave end region 4673 (“dropletvoid”) may be included at the distal end of the device. This concaveregion/void may be configured to hold a specific droplet volume, e.g.,less than a few microliters in volume (e.g., 0.1 µL, 0.2 µL, 0.3 µL, 0.4µL, 0.5 µL, 0.6 µL, 0.7 µL, 0.8 µL, 0.9 µL, 1 µL, 1.1 µL, 1.2 µL, 1.3µL, 1.4 µL, 1.5 µL, 1.6 µL, 1.7 µL, 1.8 µL, 1.9 µL, 2 µL, etc.). Largervolumes may be used as well (e.g., between 1-50 µL, between 0.1-50 µL,etc.). The droplet void region 4673 may be inserted into a solution ofthe fluid to be transferred 4675 and removed, leaving a droplet of theexpected size and volume 4677 captured within the end of the solidtransfer member 4671, as shown. This droplet may be released, e.g.,within the air gap, by immersing into a solution (e.g., another droplet)having a lower surface tension 4679 (e.g., drop gloss) causingdisplacement and release of the droplet 4677, as shown.

In general, these apparatuses may handle smaller volume droplets byincreasing the volume/amount of the immiscible fluid (drop gloss), sothat the final volume is sufficiently large for displacement within theair gap using the mechanical actuator as described herein.

FIG. 48 shows another schematic illustration of a mechanicalmicrofluidics actuator 4800. In this example the mechanicalmicrofluidics actuator includes a force applicator 4878 (e.g., stylus,bearing, roller, etc.), and a force applicator driver subassembly 4874(e.g., a force applicator subassembly). The force applicatorsub-assembly may include one or more drivers (e.g., an x and/or y motiondriver, a z-motion driver 4873, etc.), and/or a frame or gantry ontowhich the force applicator may be driven to change position and/or toapply force to the cartridge 4877 when one is seated in the cartridgeseat 4878 of the device. The force applicator sub-assembly may includeone or more stepper motors, motion rails (e.g., gantry/frame), and/orhome switches.

The mechanical microfluidics actuator of FIG. 48 also includes a thermalsub-assembly 4879 for controlling the temperature of one or more regionsof the air gap. In this example the thermal subassembly may includethermally conductive zones or regions of the seating region that may bein thermal communication with a heating and/or cooling element (e.g.,Peltier device) multiple heating/cooling elements may be included. Anyof these mechanical microfluidics actuator apparatuses 4800 may alsoinclude a magnetic control sub-assembly 4885, 4885′ for controllablyapplying a magnetic field within the air gap. FIG. 49 illustrates anexample of a row of magnetic elements (electromagnets) 4985 within thebase 4819 of a mechanical microfluidics actuator.

In some examples, the mechanical microfluidics actuator apparatus mayinclude a cartridge securement 4876, 4876′ (e.g., a holder, clamp, lock,etc.) for securing a cartridge to a cartridge seat or seating region4878 of the mechanical microfluidics actuator. In FIG. 48 , theapparatus includes a vacuum/suction sub-assembly (not shown) forapplying suction to secure the cartridge into the seating region. Insome examples the mechanical microfluidics actuator apparatus mayinclude a fluid handling (e.g., pipetting) sub-assembly 4883 for addingand/or removing fluid from the air gap. Other sub-assemblies forming apart of the mechanical microfluidics actuator apparatus may includeimaging sub-assemblies (e.g., for imaging droplets within the air gap)and/or for sensing sub-assemblies (e.g., for sensing droplets or otherinputs from the air gap and mechanical microfluidics actuator). Themechanical microfluidics actuator apparatuses described herein may alsoinclude one or more control inputs (e.g., keyboards, touchscreens,buttons, switches, etc.) and/or one or more outputs (e.g., displays,LEDs, wireless communications outputs/inputs, etc.) and hardware,software and/or firmware for controlling these. In some cases the samefeatures may be used for control inputs and outputs. In general, themechanical microfluidics actuators described herein may include one ormore controllers 4899 for controlling and coordinating operation of thevarious sub-assemblies.

Any of these apparatuses may include leveling. For example in FIG. 48the apparatus includes adjustable leveling feet 4881.

In general, the mechanical microfluidics actuators described herein maybe single cartridge use (e.g., for use with a single cartridge at atime) or may be configured to multiple-cartridge use. FIG. 50 illustratean examples of a mechanical microfluidics actuator apparatus 5001 thatis at least partially enclosed within a housing 5095 and is configuredfor use with a single cartridge 5077. The apparatus includes a forceapplicator sub-system 5097 (e.g., 3 axis motor, encoders, home and limitsensors, solenoids, raise, shafts, couplings, bearings gears, belt,etc.) and an electrical subsystem 5094 for controlling the powerrequirements of the apparatus, e.g., controller, power distribution,user-interface boards, touchscreen, etc. (and in some examples, forapplying power to one or more electrodes, e.g., for electroporationand/or electrochemical procedures on the cartridge). The apparatus alsoincludes a thermal sub-system 5092 (e.g., Peltier, high-power TECdriver, heat spreader, heat sink, etc.), a controller 5091 and aninput/output 5093 (e.g., display/touch screen). FIG. 51 illustrates anexample of an apparatus that is multiplexed 5101 to allow parallelhandling of multiple cartridges. Either the single-cartridge or amulti-configuration may also include or be configured for use with afluid handling system (e.g., liquid handler 5163) as shown in FIG. 51 .

FIGS. 52A-52B schematically illustrates one example of a mechanicalmicrofluidics actuator as described herein, showing one possiblearrangement of the subassemblies described here. For example, in FIG.52A the apparatus includes a liquid staging sub-assembly (e.g.,temperature control, inputs, tip repository, tip waste, etc.) foradding/removing liquid from the cartridge, as well as a chassissub-assembly (e.g., chassis, fans, leveling feet, switches/buttons,touchscreen, etc.) and a power distribution sub-assembly (mains powersupply, power distribution circuitry, etc.). The controller (primarycontrol PCBA) may also include sensors (e.g., ambient temperaturesensor, ambient humidity sensors, level sensors) and Wi-Fi or otherinputs/outputs. The controller may receive input from the chassissubassembly and may output/control all of these sub-assemblies. Inparticular, the controller may control the liquid handler sub-assembly,which includes motion controllers (drivers, position sensors, etc.), andmay control the sub-module with the temperature sub-assembly (Peltiersub-assembly), magnetic sub-assembly, linear motion sub-assembly andcartridge receptacle (e.g., cartridge datum) each of which may provideinput to the controller.

EXAMPLES

FIGS. 5A-53C illustrate one example of a method of moving an aqueousdroplet as described herein. In this example one or more microfluidicdroplets is manipulated so that it may be moved virtually anywhere inthe air gap 5321 formed between the first elastic sheet 5307 and asecond sheet 5309. The second sheet may also be an elastic sheet asdescribed above. Both the first 5391 and second 5392 surfaces of thefirst and second sheets (which may be referred to as the inner surfacesfacing the air gap) may be hydrophobic and oleophobic. The sheets may beformed of a hydrophobic and oleophobic material, or they may be coatedwith a hydrophobic and oleophobic material.

An aqueous fluidic droplet 5312 may be introduced into the air gapformed between the first sheet 5307 having a first surface that ishydrophobic and oleophobic and the second sheet 5309 having a secondsurface that is hydrophobic and oleophobic. As described above, thefirst sheet and the second sheet may be secured opposite andapproximately parallel at a predetermined distance relative to eachother with an air gap therebetween. The first sheet and/or the secondsheet may be held in tension. At least the first sheet is formed of anelastomeric material so that it may deform when a force (e.g., amechanical stylus, as shown in FIGS. 53B and 53C) is driven against it,will return to the approximately parallel configuration when the forceis released. For example, in FIG. 53A the mechanical force applicator(stylus 5375) is positioned above the top of the first (e.g., upper)elastomeric sheet 5307. As described above, one or more movement drives(e.g., x, y stage/z-motion, robotic stage or control) may be used todrive the movement of the mechanical force applicator relative to theupper sheet. The first and second sheets may be part of a cartridge thatmay include one or more tensioners (e.g., tensioning frames, etc.)holding the first and/or second sheets in tension.

As shown in FIG. 53B, the mechanical force actuator 5375 may be drivendown against the first sheet to a region that is adjacent to thedroplet. Locally reducing the height of the air gap (in a continuousgradient, as shown) may cause the droplet to be driven by the resultingincreased capillary force into the lower-height region. Thus, as themechanical force actuator is driven across the top of the sheet (andagainst the top sheet), as shown by FIGS. 53B and 53C causes the dropletto move within the air gap.

In any of these examples the height of the air gap may be reduced in agradient and the distance between the upper and lower (first and second)sheets is reduced but do not contact each other. For example the heightis reduced by between about 5% and 90% (e.g., between about 10% and 80%,between about 20% and 60%, between about 10% and 50%, etc.). In somecases it may be advantageous to reduce the height by between about 5%and 60%, but not more than 60% (e.g., not more than 55%, not more than50%, not more than 45%, not more than 40%, not more than 35%, not morethan 30%, not more than 25%, etc.). This may allow the gradient to drivemovement but may limit the region to a region that is local to thedroplet. This may allow the first (upper) elastic sheet to be restoredto a parallel configuration (as shown in FIG. 53C, in the regions wherethe stylet has moved away from the sheet). As shown in FIGS. 53B-53C,applying force (by the mechanical force applicator/stylet) toelastically deform the first sheet reduces the distance of the air gapbetween the first sheet and the second sheet in a local region withinthe air gap that is adjacent to the fluidic droplet, and causes thedroplet to move within the air gap, following the reduced height regionformed by the stylet.

In any of the methods and apparatuses described herein the sheetsforming the air gap are made of a hydrophobic and oleophobic material;non-hydrophobic and oleophobic materials did not work in many of theexamples shown. In addition, the materials forming the inner surface ofthe air gap may be substantially non-porous.

As mentioned, any of the droplets may be coated with a layer of dropgloss, e.g., a gloss coat that may be a low surface-tension material(e.g., oil), and may be immiscible with the droplet, which may alsoprevent or limit evaporation.

In general, the methods and apparatuses described herein may include theuse of at least 0.01% of a surfactant in or surrounding the dropletbeing moved. Surprisingly, the inventors have found that the use ofsurfactant in the droplet (e.g., 0.01% or more, 0.02% or more 0.025% ormore, between 0.01% and 1%, between 0.01% and 0.7%, between 0.01% and0.5%, between 0.01% and 0.25%, between 0.01% and 0.1%, etc.) or in agloss layer surrounding the droplet may allow the droplet to move morepredictably within the air gap when pulled by the reduced gap width asdescribed herein. Without being bound by theory, this may be due to theeffective surface tension of the droplet; the use of a surfactant ineither or both the drop gloss and/or the droplet may therefore allow thedroplet to move predictably and robustly. Without the use of asurfactant, the droplet movement may be less predictable and maysometime fail to follow the mechanical actuator as it moved across thesurface. Any appropriate surfactant may be used. For example, the dropgloss used may include a nonionic surfactant (e.g., Brij-35) or otherhydrophobic polymer. In some examples the droplet may include asurfactant such as pluronics, Tween-20, Tetronic, etc.). Thus, in any ofthese methods an apparatuses, either or both the drop gloss and/or thedroplet may include a surfactant (e.g., 0.01% or more surfactant). Insome cases the surfactant may be added before beginning any of the stepinvolving moving the droplet by locally reducing the gap width in theregion adjacent to the droplet.

In examples in which a mechanical force applicator (e.g., stylus) isused, the contact surface of the stylus may be sized proportional to theair gap and/or the droplet size/volume. In particular, the aspect ratioof the stylus, e.g., the size of the stylus tip relative to the size ofthe droplet, and/or the size of the stylus tip relative to the height ofthe air gap, may be selected to be between 1:0.5 and 1:20 (tip:droplet).

DNA Sequencing and DNA Synthesis

The methods and apparatuses described herein may be used specificallyfor performing enzymatic process on polynucleotides, including (but notlimited to) sequencing and/or synthesis.

For example, these methods and apparatuses may be used to perform DNASequencing-By-Synthesis (SBS). SBS provides many significant benefits tothe scientific research community and has enabled many new diagnosticapplication, including an increase in the output by sequencinginstrumentation, faster turnaround time for results and the reduction ofcosts by orders of magnitude versus the prior dominant sequencingmethodology, Sanger Sequencing. Sanger Sequencing relies onelectrophoretic separation of DNA fragments created by speciallymodified terminating nucleotides. SBS eliminates the requirement for theseparation and allows the implementation of massively parallelsequencing approaches. Several important clinical applications have beendeveloped as a result of these cost and throughput improvementsincluding, Non-Invasive Pre-Natal Testing (NIPT) to detect aneuploidiessuch as Down’s Syndrome in pregnant women’s blood, genetic carriertesting to provide information to parents regarding potential geneticrisks, oncology patient stratification, tumor profiling and earlydetection of cancer through the sequencing of nucleic acids in blood(known as cell-free sequencing).

SBS is a flow-based sequencing technique in which a series of liquidformulations are introduced to a flow cell populated with DNA templatesisolated, purified and processed to create a sequencing “library” andflowed into the sequencing flow cell. The flow cell is loaded withtemplate DNA onto either random or structured arrays which create adistinct “cluster” for each DNA library fragment. Reagents are deliveredin a sequential process which enzymatically adds a single fluorescentlylabeled nucleotide per cycle. After each fluorescent nucleotideaddition, the flow cell is imaged, with each unique fluorescent labelsrepresenting a specific base (A, C, G, T) and processed through softwarewhich assigns the next base in the sequence. After imaging, thefluorescent dye and the nucleotide blocking group are chemically cleavedand washed away to prepare for the next cycle. Typically, this processis repeated for 75-600 cycles adding another base each cycle which iscaptured through the imaging process and software analysis. Manyprotocols have a process (mid-run in most cases) to create acomplementary strand of DNA which is also sequenced to improve coverageand accuracy. The method for creating this complementary strand usessimilar reagents to create newly synthesized DNA to be sequenced by SBS.

Current systems typically use a delivery method to provide thecontinuous flow of each reagent sequentially. These are delivered by apumping or pressure mechanism which floods the flow cell with eachreagent completely filling the flow cell then flushed and replaced withthe next reagents required to drive the cycle. While these volumes arefairly small, they do require excess volumes to ensure no carryover fromstep to step or cycle to cycle which could compromise the resultingsequencing. FIG. 54 schematically illustrates a sequencing cycle andoverall process map for SBS. The microfluidic methods and apparatusesdescribed herein may be used to flow the various components within the‘flow cell’ configured as described herein, including the wash steps.

Although traditional digital microfluidics (e.g., using electrowetting,etc.) has been proposed for use in sample preparation of nucleic acidsas a front end to the sequencing process and as an alternative to manualbenchtop library creation or conventional robotic pipetting systems,there are a number of drawbacks. Electro-wetting on Dielectric (EWOD)has been successfully applied to the front-end processes such as theisolation of DNA from patient samples and the creation of a sequencinglibraries to be subsequently loaded on a sequencing instrument. Whileelectrowetting has a reasonable fit for the automation of up-frontprocesses, the complexities of the sequencing process itself presentseveral technical and practical economic challenges. Of particular noteare the requirements for imaging the flow cell after each nucleotideaddition cycle. In an ideal implementation, a completely integratedsequencing processes would allow a sample to be introduced to a systemand would provide DNA sequencing results as the output. EWOD is unlikelyto be successful as a fluidic solution for such a fully integratedprocess.

The methods and apparatuses described herein, which may be referred toas mechanical actuation on the surface, in which a mechanical force(e.g., compressive force) is applied to drive one or more droplets mayprovide significant advantages as compared to other microfluidictechniques, including electrowetting, and may allow for the possibilityof using a single fluidic technology across the entire sequencingprocess, including SBS. As described above, the use of a mechanicalcompression to change the capillary force to move droplets in twodimensions may have many advantages, particularly in regards to samplepreparation, and may be used for virtually all of the necessary steps,such as nucleic acid isolation, library generation, cluster generation,primer loading and hybridization, and multi-cycle sequencing reactions,including the steps illustrated in FIG. 54 .

FIGS. 55A-55B illustrate an example of the use of mechanical compressionto change the capillary force in order to move droplets within a flowcell 5501 to introduce sequencing primers to clusters of DNA templates(e.g., part of the sequencing by synthesis process described above).FIG. 55A, shows the loading and wash steps associated with primerintroduction to the sequencing templates in the flow cell 5501. As inany of the methods described herein, multiple mechanical forceapplicators (styluses 5575) may be used concurrently on the same flowcell (e.g., cartridge). These mechanical force applicators may beindependently or collectively controlled/actuated. In FIG. 55A, thecartridge/flow cell includes a region in which primers to which clustershave been formed are arranged, either in an un-patterned or patterned(e.g., in nanowells) arrangement. The steps for generating the clustersmay also be performed using the mechanical force applicator as describedherein, or they may be performed by pipetting and washing.

The first droplet 5502 is drawn onto the clusters first using the firststylet and may be allowed to incubate over the clusters (allowinghybridization), as shown in FIG. 55B, and may then be drawn off, and insome cases out of the flow cell (e.g., to a waste depot). The seconddroplet 5504 (wash buffer) may then be drawn over the clusters to washthe clusters.

FIGS. 56A-56E illustrate the steps associated with each cycle of SBS, asperformed in a cartridge (e.g., flow cell) 5501. As described above,either multiple mechanical force applicators (styluses 5575), or thesame stylus may be used sequentially. In this examples, the figures showone direction of movement of droplets, e.g., from left to right.However, the methods and apparatuses described herein may allow movementin two dimensions (e.g., in the entire plane of the air gap, and mayallow movement in any arbitrary direction in this plane). Thus, in someexamples the reagent droplets may be moved to the side and reintroducedand or reused (and even recharged with depleted components, e.g.,nucleotides, enzymes, other chemicals).

In FIG. 56A, following primer hybridization and washing (see FIGS.55A-55B), may be followed by the SBS cycles of applying nucleotide,polymerase, reaction mix, washing, and extension with imaging formultiplexed sequencing. FIG. 56A shows the start of the first cycle, inwhich nucleotides and polymerase are added to the clusters in the airgap of the cartridge/flow cell 5501 by pulling the droplet including thenucleotide and polymerase using a mechanical force actuator. After anappropriate time, the droplet may be pulled off of the clusters and oneor more wash droplets may be moved onto the clusters (FIG. 56B) whileimaging (to identify the additional nucleotide). The wash droplet mayagain be moved off of the clusters and a droplet includingdye/terminator cleavage components may be pulled onto the clusters (FIG.56C); this droplet may then be moved off using the mechanical forceactuator and the same or a different mechanical force actuator may beused to move another droplet of nucleotides and polymerase for the startof the second cycle (FIG. 56E).

The microfluidic methods and apparatuses described herein may also beused for other applications, in addition to SBS Sequencing. For example,these methods and apparatuses may be used for enzymatic synthesis of DNAoligos (which is very similar to the SBS process), such as cyclicalenzymatic addition of nucleotides with reversible terminators. Othernonlimiting examples may include DNA oligo synthesis.

For example, the methods and apparatuses described herein may be usedfor nucleic acid extraction (illustrated in FIGS. 57 and 58A-58B) andlibrary preparation (FIG. 59 , FIG. 60 , and FIG. 61 ). FIG. 57schematically illustrates an overall workflow from nucleic acidextraction to sequencing using a system for mechanical actuation on thesurface (e.g., mechanical compression to change the capillary force).

In FIG. 57 , a tope and side view of an 8-lane cartridge similar tothose described above may be integrated to a system containing an arrayof cooling regions (e.g., Peltier) and magnetic and/or resistive heatingzones that may allow the cartridge and system to perform DNA/RNAextraction, library preparation, and sequencing at specified regions. InFIG. 57 the cartridge includes an air gap with a plurality of differentreaction wells (including thermal control/cooling). The cartridge isdivided into lanes (8 lanes are shown) and includes regions for DNA/RNAsequencing, Library preparation and sequencing (e.g., SBS). Lanedividers may divide the lanes.

FIG. 58 illustrates the operation of the cartridge and system shown inFIG. 57 for polynucleotide (e.g., DNA, RNA, etc.) extraction from aclinical sample. FIG. 58A shows a side-view schematic of DNA/RNAextraction from a clinical samples on the cartridge. In this example adroplet of a 250 µl clinical sample (e.g., blood, saliva and tissuehomogenate, etc.) containing 45 µl of dropgloss and a 310 µL droplet oflysis buffer are merged and mixed by the methods described herein for 5min in the PCR reaction well and incubate at 70° C. for 10 minutes. Thereaction (Rxn) droplet is then actuated to the Magnet/Resistive Heaterzone, merged with 400 µL HDQ Binding Buffer and 20 µL Mag-Bind®Particles HDQ, and mixed for 10 minutes; the magnet is then engageduntil pellet formation, supernatant is discarded to waste, and thepellet is washed twice with 600 µL VHB wash Buffer. The pellet may beresuspended in 600 µL SPM wash Buffer and driven by mechanical actuationon the surface to clean the Magnet/Resistive Heater zone, the magnet maybe engaged, and the wash buffer may be moved to waste. Finally, theelute nucleic acid is eluted in 30 µ1 of Eluent.

In FIG. 58 , the droplet of clinical sample (e.g., blood, saliva andtissue homogenate) merges with the lysis buffer 581 and gets mixed bythe techniques described herein (e.g., mechanical actuation on thesurface) for 5 min in the reaction well and then incubates at 70° C. for10 minutes. The lysate droplet is then actuated to the Magnet/ResistiveHeater zone 582, merged with Binding Buffer and Magnetic/Binding beadparticles and may be mixed for 10 minutes. The magnet may then beengaged until pellet formation 583, the supernatant discarded to waste,and the pellet may be washed twice with wash Buffer. The pellet isresuspended in wash Buffer and driven by mechanical actuation at thesurface to clean the Magnet/Resistive Heater zone, the magnet may beengaged, and the wash buffer may be moved to waste. Finally, the elutionbuffer gets actuated to the pelleted beads to elute nucleic acid offbeads.

FIG. 59 illustrates an RNA sequencing workflow. In FIG. 59 , a side-viewthrough the cartridge shows a schematic of RNAseq workflow on thecartridge. At step 591, 2 ul droplet of fragmented RNA andfirst-stranded synthesis master mix with 45 µL of dropgloss droplet areactuated by mechanical actuation at the surface, as described herein(e.g., using a stylus) to the PCR Reaction Well and incubated (25° C.for 10 min, 42° C. for 15 min, 70° C. for 15 min). Thereafter, 6 µldroplet of second-strand synthesis master mix is added to the reaction(Rxn) droplet and incubated at 16° C. for 60 min. The Rxn droplet isactuated to the Magnet/Resistive Heater zone 592, merged with 11.2 ulbead droplet, mixed and incubated for 5 min at RT, the magnet is engageduntil pellet formation, the supernatant is discarded to waste, thepellet is washed twice with 25 ul with 80% EtOH (not shown inschematic), and the cDNA sample is eluted off beads into 6 µl elutionbuffer. Afterwards, 5 ul droplet of cDNA sample is then actuated asdescribed herein to the PCR Reaction Well, merged with 1 µl of end prepmaster mix (with 10 µl dropgloss), and incubated at 20° C. for 30 minfollowed by 65° C. for 30 min. Then, 3.1 µl of adaptor ligation mastermix and 0.25 µl of adaptors droplets are actuated as described herein,mixed with Rxn droplet and incubated at 20° C. for 15 min 593. Theadapter ligation Rxn droplet is actuated to the Magnet/Resistive Heaterzone, merged with 7.28 µl of bead droplet, mixed and incubated for 5 minat RT, and the magnet is engaged until pellet formation, after whichsupernatant is discarded to waste, the pellet is washed twice with 25 ulwith 80% EtOH (not shown in schematic), and the DNA library is eluted in6 µl in nuclease-free water containing 5 uM TRUESEQ BARCODES 594.

5 ul droplet of purified DNA library sample may then be actuated bymechanical actuation (e.g., using a stylus) to the PCR Reaction Well,merged with 10.9 µl of USER/PCR master mix (with 45 µl dropgloss), andincubated at 37° C. for 15 min, 98° C. for 30, and then cycled 19X at98° C. for 10 sec, 65° C. for 75 sec 595. The amplified DNA droplet isactuated to the Magnet/Resistive Heater zone, merged with 12.72 µl ofbead droplet, mixed and incubated for 5 min at RT, a magnet is engageduntil pellet formation, the supernatant is discarded to waste, thepellet is washed twice with 25 ul with 80% EtOH (not shown inschematic), and the DNA library is eluted in 25 µl of Eluent 596.

In FIG. 59 , the droplet of fragmented RNA and first-stranded synthesismaster mix with dropgloss are actuated by mechanical actuation (e.g.,stylus) to the Reaction Well and incubated (25° C. for 10 min, 42° C.for 15 min, 70° C. for 15 min) 591. Then a droplet of second-strandsynthesis master mix is added to the reaction (Rxn) droplet andincubated at 16° C. for 60 min. The Rxn droplet is actuated to theMagnet/Resistive Heater zone, merged with SPRI or Ampure beads droplet,mixed and incubated for 5 min at room temperature (RT), magnet engageduntil pellet formation, supernatant discarded to waste, pellet washedtwice with 80% EtOH (not shown in schematic), and cDNA sample eluted offbeads into elution buffer 592. The droplet of cDNA is then actuated(e.g., using a stylus) to the Reaction Well, merged with end prep mastermix and incubated at 20° C. for 30 min followed by 65° C. for 30 min593. Then adaptor ligation master mix and adaptors droplets are actuatedby mechanical actuation (e.g., stylus), mixed with Rxn droplet andincubated at 20° C. for 15 min. The adapter ligation Rxn droplet isactuated to the Magnet/Resistive Heater zone, merged with SPRI or Ampurebead droplet, mixed and incubated for 5 min at RT, magnet engaged untilpellet formation, supernatant discarded to waste, the pellet washedtwice with 80% EtOH (not shown in schematic), and DNA library eluted innuclease-free water containing primers 594. The purified DNA libraryplus primers mix is mechanically actuated as described herein (bystylus) to the PCR Reaction Well, merged with USER/PCR master mix withdropgloss and incubated at 37° C. for 15 min, 98° C. for 30, and thencycled up to 19X at 98° C. for 10 sec, 65° C. for 75 sec 595. Theamplified DNA droplet is actuated to the Magnet/Resistive Heater zone,merged with SPRI/Ampure bead droplet, mixed and incubated for 5 min atRT, magnet engaged until pellet formation, supernatant discarded towaste, pellet washed twice with 80% EtOH (not shown in schematic), andRNA-seq library eluted in 25 µl of elution buffer 596.

FIG. 61 is a schematic side-view of a second part of a twist exometarget enrichment methods using a cartridge as described herein. In thisexample, an 8.3 µl droplet of DNA and hybridization mix a with 45 µL ofdropgloss droplet are actuated as described herein to the PCR ReactionWell and incubated at 95° C. for 5 min and 60C for 2 hours 601. The Rxndroplet is actuated to the Magnet/Resistive Heater zone, merged with33.3 µl Streptavidin beads, mixed for 30 min at RT, magnet engaged untilpellet formation, supernatant discarded to waste, pellet is initiallywashed with a 70° C. pre-heated 50 µl buffer droplet followed by a 48°C. pre-heated 50 µl buffer droplet (not shown in schematic), andpurified DNA library sample eluted off beads into 7.5 µl elution buffer602. Thereafter, 7.5 µl droplet of purified DNA library sample is thenactuated as described herein to the PCR Reaction Well, merged with 0.83µl Primer and 8.3 µl of master mix (with 10 µl dropgloss), and incubatedat 97° C. for 45 sec followed by 8 cycles at 97° C. for 15 s, 60 for 30s, 72C for 30 s, and finally 72C for 1 min 603. The amplified DNAdroplet is actuated to the Magnet/Resistive Heater zone, merged with 30ul magnetic bead droplet, mixed and incubated for 5 min at RT, magnetengaged until pellet formation, supernatant discarded to waste, pelletwashed twice with 25 µl with 80% EtOH (not shown in schematic), and DNAlibrary eluted in 30 µl of Eluent 604.

Thus, in FIG. 60 the method for twist exome target enrichment isperformed on a cartridge as described herein, using mechanical actuationof the surface of the cartridge. In FIG. 60 the droplet of DNA andhybridization mix with dropgloss 601 and are actuated as describedherein to the Reaction Well and incubated at 95° C. for 5 min and 60Cfor up to 4 hours. The Rxn droplet is actuated to the Magnet/ResistiveHeater zone, merged with Streptavidin beads, mixed for 30 min at RT,magnet engaged until pellet formation, supernatant discarded to waste,pellet is initially washed with a 70° C. pre-heated buffer dropletfollowed by a 48° C. pre-heated 50 µl buffer droplet (not shown inschematic), and purified DNA library sample eluted off beads intoelution buffer 602. The droplet of purified DNA library sample is thenactuated by the mechanical actuation of the surface as described hereinto the PCR Reaction Well, merged with Primers and dropgloss andincubated at 97° C. for 45 sec followed up to 18 cycles at 97° C. for 15s, 60 for 30 s, 72C for 30 s, and finally 72C for 1 min 603. Theamplified DNA droplet is actuated to the Magnet/Resistive Heater zone,merged with SPRI/Ampure magnetic bead droplet, mixed and incubated for 5min at RT, magnet engaged until pellet formation, supernatant discardedto waste, pellet washed twice with 80% EtOH (not shown in schematic),and DNA library eluted in Elution buffer 604.

FIG. 61 illustrates a workflow showing Aplicon-seq. For example, FIG. 61shows a side-view schematic of an Ampliseq (2 primer pools) workflow ona cartridge as described herein. First, three droplets of 13.5 µl DNA, 9µl HiFi mix and 22.5 µl water are merged by mechanical actuation of thesurface (e.g., using a stylus), mixed for 5 sec at RT, split into twoequal droplets using a liquid handler (not shown in schematic) 611.Second, each of the droplets were merged with a 5 µl unique primerdroplets with 45 µL of dropgloss droplet, and Rxn droplets (1&2)actuated by mechanical actuation of the surface (e.g., using a stylus)to the PCR Reaction Well zones and incubated (99° C. for 2 min, and then17 cycle: at 99C for 15 s, 60C for 4 min) 611′. Next, Rxn droplets 1 &2are merged by mechanical actuation of the surface (e.g., using astylus), actuated to the Magnet/Resistive Heater zone, merged with 4 µlFuPa reagent droplet, mixed and incubated at 50° C. for 10 min, 55C for10 min and 60C for 20 min. Second, 8 µl Switch solution, 4 µl Barcodeadaptor mix and DNA Ligase droplets were added to the Rxn droplet bymechanical actuation of the surface (e.g., using a stylus) and incubatedat 22° C. for 30 min, 68C for 5 min, and 72C for 5 min 612. Afterwards,90 µl droplet of beads were added to the Rxn droplet, mixed andincubated for 5 min at RT, magnet engaged until pellet formation,supernatant discarded to waste, pellet washed twice with 150 ul of 80%EtOH droplets (not shown in schematic), and library eluted off beadsinto 50 µL of Platinum™ PCR SuperMix HiFi and 2 µL of Equalizer™ Primersdroplets.

In 613, 50 µl droplet of purified library is actuated by mechanicalactuation of the surface (e.g., using a stylus) to the PCR ReactionWell, and incubated at 98° C. for 2 min and cycled 9 times at 98C for 15s, 64C for 1 min. Second, 10 µL of Equalizer Capture droplet is added tothe Rxn droplet and mixed for 5 min at RT. In 614, the Rxn droplet isactuated to the Magnet/Resistive Heater zone, merged with 6 µL of washedEqualizer™ Beads, mixed and incubated for 5 min at RT, magnet engageduntil pellet formation, supernatant discarded to waste, pellet washedtwice with 150 µl with 80% EtOH (not shown in schematic), and DNAlibrary eluted in 100 µl of Eluent droplet.

Thus, as shown in FIG. 61 , the Ampliseq (2 primer pools) workflow, in611, three droplets of DNA, PCR mastermix and water are merged bymechanical actuation of the surface (e.g., using a stylus), mixed for 5sec at RT, split into two equal droplets using a liquid handler (notshown in schematic). Second, 611′ each of the droplets were merged witha unique primer droplet with dropgloss, and Rxn droplets (1&2) actuatedby mechanical actuation of the surface (e.g., using a stylus) to the PCRReaction Well zones and incubated (99° C. for 2 min, and then 17 cycle:at 99C for 15 s, 60C for 4 min). In step 612, first, Rxn droplets 1 &2are merged by mechanical actuation of the surface (e.g., using astylus), actuated to the Magnet/Resistive Heater zone, merged with FuPareagent droplet, mixed and incubated at 50° C. for 10 min, 55C for 10min and 60C for 20 min. Second, Switch solution, Barcode adaptor mix andDNA Ligase droplets were added to the Rxn droplet by mechanicalactuation of the surface (e.g., using a stylus) and incubated at 22° C.for 30 min, 68C for 5 min, and 72C for 5 min. Third, droplet of beadswere added to the Rxn droplet, mixed and incubated for 5 min at RT,magnet engaged until pellet formation, supernatant discarded to waste,pellet washed twice with 80% EtOH droplets (not shown in schematic), andlibrary eluted off beads into Platinum™ PCR SuperMix HiFi and Equalizer™Primers droplets. In 613, first, 50 ul droplet of purified library isactuated by mechanical actuation of the surface (e.g., using a stylus)to the PCR Reaction Well, and incubated at 98° C. for 2 min and cycled 9times at 98C for 15 s, 64C for 1 min. Second, Equalizer Capture dropletis added to the Rxn droplet and mixed for 5 min at RT. As 614, The Rxndroplet is actuated to the Magnet/Resistive Heater zone, merged withwashed Equalizer™ Beads, mixed and incubated for 5 min at RT, magnetengaged until pellet formation, supernatant discarded to waste, pelletwashed twice with 80% EtOH (not shown in schematic), and DNA libraryeluted in Elution droplet.

Introducing and Removing Liquid

FIGS. 62A-62J illustrate one example of a method of applying liquid(droplets) into a cartridge such as the cartridges described herein. Forexample, in FIG. 62A, a partial section through the cartridge shows anopening into which a pipette tip may be inserted. A standard pipette tipmay be used. As an initial step, a droplet of drop gloss material (asdescribed above) may be pipetted into the air gap of the cartridge. Forexample, between about 10-45 µL of drop gloss may be pipetted into theair gap, and the pipette tip removed (FIG. 62B). A droplet of theaqueous reaction material may then be inserted into the air gap in thesame way, as shown in FIG. 62C. The droplet may be pipetted onto, intoor adjacent to the drop gloss (which is added first). In some examples,the drop gloss may be combined with the droplet before they are pipettedtogether. Alternatively, the drop gloss may be added after the aqueous(reaction) droplet is added. In general, the liquid material may beintroduced by pipette tip, using a unique (dedicated single tip/sample)application or universal (shared tip for multi-dispense) application ofreagents across one or more lanes of the cartridge. In FIG. 62C anyvolume of aqueous reaction mixture may be used, such as between about250 nL to 80 µL.

In FIGS. 62C-62D, pre-dispensed drop gloss encapsulates aqueous reagentand protects from surface fouling and evaporation during workflow steps.Ethanol and wash buffers do not need drop gloss. The volume of dropgloss may be more than, less than or the same as the volume of theaqueous droplet. In some example, as shown in FIGS. 62A-62D, the volumeof the drop gloss may exceed the volume of the reaction droplet (e.g.,by more than 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, between 1 and 10times, between 1 and 8 times, between 1 and 7 times, between 1 and 6times, between 1 and 5 times, between 1 and 4 times, etc. or more).Thus, very small volumes of reaction droplet (e.g., as small as 250 nL)can be manipulated; the reaction droplet may be combined with an excessof drop gloss which may encapsulate it and allow it to be manipulated asdescribed herein (moved, combined, split, heated, mixed, etc.) even inchannels having relatively large channel heights (e.g., 1.5 mm or more).Thus, in some examples the systems described herein can dispense as lowas 250 nL and as high as 80 uL of reagents/mastermix/sample volumes. Insome examples, for drop gloss the system can dispense 10-45 uL volumes.The lane width illustrated in some of these examples can accommodate upto 150 uL total volume (drop gloss + reagent) or up to 80 uL reagentvolume. Larger or smaller lane widths and/or heights may be used.

As shown above, during introduction of liquids into each lane’s inlet,the dispensing tip is lowered (straight down) against the bottom filmsurface in a position to ensure that part of the droplet is inside thechannel (due to the innate wetting properties of liquids) whendispensed. Capillary pressure may draw the droplet into the air gap andaway from the opening (or to the edge of the opening) so that it can bemanipulated, as shown in FIGS. 62E-62G. Thus, for small volumes such as250 nL, a pre-dispensed carrier dropgloss droplet (e.g., ~5-10 ul) maybe used into which the 250 nL droplet is dispensed, and then acompression force is applied to the one-side of the port pulling in thecarrier drop gloss droplet containing the small volume. For example, asshown in FIGS. 62E-62F, the mechanical manipulator (stylus) may belowered onto the elastically deformable upper sheet to reduce the height(either on or more preferably adjacent to the droplet) and themechanical manipulator (stylus) may be drawn across the surface of theupper sheet as shown in FIG. 62G to move the droplet, which issurrounded by the drop gloss. As shown, the reagent is protected insideof the drop of drop gloss. In FIG. 62F, the stylus compresses the filmsurface adjacent to the inlet hole (at a safe distance that will avoidcontamination of the stylus). The droplet of drop gloss/reagent is thendrawn into the narrower gap (by the capillary action, including theincrease in capillary force) and the droplet is now fully inserted tothe lane and sandwiched across its surface between a top and bottomfilm. The stylus will keep driving 2-phase mix (e.g., drop gloss andaqueous droplet) across the heating/cooling and/or magnet/isothermalheater zones to conduct different protocol steps.

FIGS. 62H to 62J illustrate removal of the droplet from the cartridge.The drop gloss material may be first removed from the droplet, e.g., bycontacting an oleophilic material that may wick off the material, bymechanical separation, etc. Alternatively, the droplet may contain thedrop gloss with the aqueous material. In FIG. 62H, the stylus drivesreaction droplet (e.g., containing a product library, or other reactionproduct, as illustrated and described above) adjacent to the openingthrough the upper sheet into the air gap (e.g., an inlet hole, etc.) ata safe distance that will avoid contamination of the stylus. This isillustrated in FIG. 62I. The droplet to be removed is near but not inthe opening into the air gap. However, because the upper sheet is formedof an elastic material, it may be deformed by the pipette top foraccess, as shown in FIG. 62J. In this example, the pipette tip isinserted to the inlet hole (FIG. 62I) and reaches a position over thebottom film. It then gets moved towards the droplet (FIG. 62J),temporarily deforming the top sheet (film) until it reaches sampleposition (the droplet is now only partially sandwiched between a top andbottom sheets) and the droplet is aspirated up into the pipette untilfully removed or until a specific volume is removed. The pipette tip maythen move back to inlet hole opening and then elevates to travel toproduct destination (e.g., a tube/plate, etc.) so that the operator maycollect the material at end of run.

Evaporation Control

In general, these methods and apparatuses may be configured to preventor reduce evaporation. In general, drop gloss coatings of the aqueousmaterial, alone or in combination with the application of force (e.g.,mechanical force) against the droplet may both enhancing uniformity ofheating and/or may prevent evaporation. For example in some variationsof the methods and apparatuses described herein, the aqueous droplet mayexperience less than 10% evaporation (e.g., less than 9%, less than 8%,less than 7%, less than 6%, etc.) evaporation when heated to 95° C. orhigher for at least 30 minutes. In one example, a droplet of aqueousreaction mixture heated to 95° C. for 30 min (20 µL in 45 µL drop gloss)experienced approximately 5.8% evaporation in total.

FIG. 63 illustrates an example in which the droplet (e.g., drop glossplus aqueous droplet) were held in a reaction well formed in the bottomlayer by applying suction to conform the bottom layer (which, like thetop layer, is elastically deformable) to form a well in the air gap. Thebase of the drive system holding the cartridge, including the seatingregion, is therefore shaped to form the well as the bottom layer isattached via suction to the seating region. This well is also a thermalcontrol region including a heater for controlling the temperature of thedroplet in the air gap. In FIG. 63 the droplet was heated to 95° C. for40 min (20 µL in 45 µL drop gloss). The stylet (shown in this example asa roller stylus) may be held over the top of the sheet, over thedroplet. This applied mechanical force may hold or pin the droplet inposition relative to the heating region. This may also help thermallyinsulate the droplet. In some examples the portion of the stylet overthe droplet may be a thermal insulating material. In some examples thelower layer (sheet) may be more thermally permissive than the upperlayer (sheet).

Thus, even as compared to other microfluidic systems, the methods andapparatuses described herein may prevent evaporation in the air gapsurprisingly well.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein and may be used toachieve the benefits described herein.

The process parameters and sequence of steps described and/orillustrated herein are given by way of example only and can be varied asdesired. For example, while the steps illustrated and/or describedherein may be shown or discussed in a particular order, these steps donot necessarily need to be performed in the order illustrated ordiscussed. The various example methods described and/or illustratedherein may also omit one or more of the steps described or illustratedherein or include additional steps in addition to those disclosed.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like. For example, any of the methodsdescribed herein may be performed, at least in part, by an apparatusincluding one or more processors having a memory storing anon-transitory computer-readable storage medium storing a set ofinstructions for the processes(s) of the method.

While various embodiments have been described and/or illustrated hereinin the context of fully functional computing systems, one or more ofthese example embodiments may be distributed as a program product in avariety of forms, regardless of the particular type of computer-readablemedia used to actually carry out the distribution. The embodimentsdisclosed herein may also be implemented using software modules thatperform certain tasks. These software modules may include script, batch,or other executable files that may be stored on a computer-readablestorage medium or in a computing system. In some embodiments, thesesoftware modules may configure a computing system to perform one or moreof the example embodiments disclosed herein.

As described herein, the computing devices and systems described and/orillustrated herein broadly represent any type or form of computingdevice or system capable of executing computer-readable instructions,such as those contained within the modules described herein. In theirmost basic configuration, these computing device(s) may each comprise atleast one memory device and at least one physical processor.

The term “memory” or “memory device,” as used herein, generallyrepresents any type or form of volatile or non-volatile storage deviceor medium capable of storing data and/or computer-readable instructions.In one example, a memory device may store, load, and/or maintain one ormore of the modules described herein. Examples of memory devicescomprise, without limitation, Random Access Memory (RAM), Read OnlyMemory (ROM), flash memory, Hard Disk Drives (HDDs), Solid-State Drives(SSDs), optical disk drives, caches, variations or combinations of oneor more of the same, or any other suitable storage memory.

In addition, the term “processor” or “physical processor,” as usedherein, generally refers to any type or form of hardware-implementedprocessing unit capable of interpreting and/or executingcomputer-readable instructions. In one example, a physical processor mayaccess and/or modify one or more modules stored in the above-describedmemory device. Examples of physical processors comprise, withoutlimitation, microprocessors, microcontrollers, Central Processing Units(CPUs), Field-Programmable Gate Arrays (FPGAs) that implement softcoreprocessors, Application-Specific Integrated Circuits (ASICs), portionsof one or more of the same, variations or combinations of one or more ofthe same, or any other suitable physical processor.

Although illustrated as separate elements, the method steps describedand/or illustrated herein may represent portions of a singleapplication. In addition, in some embodiments one or more of these stepsmay represent or correspond to one or more software applications orprograms that, when executed by a computing device, may cause thecomputing device to perform one or more tasks, such as the method step.

In addition, one or more of the devices described herein may transformdata, physical devices, and/or representations of physical devices fromone form to another. Additionally or alternatively, one or more of themodules recited herein may transform a processor, volatile memory,non-volatile memory, and/or any other portion of a physical computingdevice from one form of computing device to another form of computingdevice by executing on the computing device, storing data on thecomputing device, and/or otherwise interacting with the computingdevice.

The term “computer-readable medium,” as used herein, generally refers toany form of device, carrier, or medium capable of storing or carryingcomputer-readable instructions. Examples of computer-readable mediacomprise, without limitation, transmission-type media, such as carrierwaves, and non-transitory-type media, such as magnetic-storage media(e.g., hard disk drives, tape drives, and floppy disks), optical-storagemedia (e.g., Compact Disks (CDs), Digital Video Disks (DVDs), andBLU-RAY disks), electronic-storage media (e.g., solid-state drives andflash media), and other distribution systems.

A person of ordinary skill in the art will recognize that any process ormethod disclosed herein can be modified in many ways. The processparameters and sequence of the steps described and/or illustrated hereinare given by way of example only and can be varied as desired. Forexample, while the steps illustrated and/or described herein may beshown or discussed in a particular order, these steps do not necessarilyneed to be performed in the order illustrated or discussed.

The various exemplary methods described and/or illustrated herein mayalso omit one or more of the steps described or illustrated herein orcomprise additional steps in addition to those disclosed. Further, astep of any method as disclosed herein can be combined with any one ormore steps of any other method as disclosed herein.

The processor as described herein can be configured to perform one ormore steps of any method disclosed herein. Alternatively or incombination, the processor can be configured to combine one or moresteps of one or more methods as disclosed herein.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature’s relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/- 0.1% of the stated value (or range of values), +/-1% of the stated value (or range of values), +/- 2% of the stated value(or range of values), +/- 5% of the stated value (or range of values),+/- 10% of the stated value (or range of values), etc. Any numericalvalues given herein should also be understood to include about orapproximately that value, unless the context indicates otherwise. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. Any numerical range recited herein is intended to include allsub-ranges subsumed therein. It is also understood that when a value isdisclosed that “less than or equal to” the value, “greater than or equalto the value” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that throughout the application, datais provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A method of sequencing by synthesis usingmechanical compression, the method comprising: sequentially moving eachdroplet of a series of fluidic droplets within a cartridge, wherein thecartridge comprises an air gap formed between a first sheet having afirst surface that is hydrophobic and oleophobic and a second sheethaving a second surface that is hydrophobic and oleophobic, wherein thefirst sheet and the second sheet are secured opposite each other at apredetermined spacing relative to each other with the air gaptherebetween, further comprising a plurality of clusters ofpolynucleotides on the first sheet or the second sheet, wherein movingeach droplet comprises moving one or more mechanical force applicatorsagainst the first sheet to elastically deform the first sheet and createa region of reduced spacing between the first and second sheets so thateach droplet follows the region of reduced spacing over the plurality ofclusters of polynucleotides, wherein the series of fluidic dropletsincludes: a nucleotide polymerase reaction mix droplet, a first washbuffer droplet, a dye/terminator cleavage mix droplet, and a second washbuffer droplet; and imaging the plurality of clusters of polynucleotidesto detect addition or hybridization of a nucleotide to clusters of theplurality of clusters of polynucleotides.
 2. The method of claim 1,further comprising repeating the steps of sequentially moving eachdroplet and imaging the plurality of clusters to generate sequence datafor the plurality of clusters of polynucleotides.
 3. The method of claim1, further comprising introducing the series of fluidic droplets intothe air gap.
 4. The method of claim 3, wherein sequentially moving eachdroplet comprises moving each droplet as it is introduced into the airgap.
 5. The method of claim 1, wherein imaging comprises imaging theplurality of clusters of polynucleotides after moving the first washbuffer droplet over the plurality of clusters of polynucleotides.
 6. Themethod of claim 1,wherein the plurality of clusters of polynucleotidesare arranged in an un-patterned arrangement.
 7. The method of claim 1,wherein moving the one or more mechanical force applicators against thefirst sheet to elastically deform the first sheet comprises moving oneor more styluses against the first sheet.
 8. The method of claim 1,further comprising repeatedly applying and releasing a compression forceto the first sheet over the plurality of clusters of polynucleotides tomix one droplet of the series of fluidic droplets over the plurality ofclusters of polynucleotides.
 9. The method of claim 1, whereinsequentially moving each droplet of the series of fluidic dropletsfurther comprises removing each droplet of the series of fluidicdroplets from the air gap after it been moved over the plurality ofclusters of polynucleotides.
 10. The method of claim 1, wherein eachdroplet of the series of fluidic droplets has a volume between 10⁻⁶ and10⁻¹⁵ liters.
 11. The method of claim 1, wherein the air gap is open toatmospheric pressure and unpressurized.
 12. The method of claim 1,further comprising hybridizing a library of polynucleotides to eitherthe first sheet or the second sheet in the air gap to form the clustersof polynucleotides.
 13. A method of sequencing by synthesis usingmechanical compression, the method comprising: sequentially moving eachdroplet of a series of fluidic droplets within a cartridge, wherein thecartridge comprises an air gap formed between a first sheet having afirst surface that is hydrophobic and oleophobic and a second sheethaving a second surface that is hydrophobic and oleophobic, wherein thefirst sheet and the second sheet are secured opposite each other at apredetermined spacing relative to each other with the air gaptherebetween, further comprising a plurality of clusters ofpolynucleotides on the second sheet, wherein moving each dropletcomprises moving one or more mechanical force applicators against thefirst sheet to elastically deform the first sheet and create a region ofreduced spacing between the first and second sheets so that each dropletfollows the region of reduced spacing over the plurality of clusters ofpolynucleotides, wherein the series of fluidic droplets includes: anucleotide polymerase reaction mix droplet, a first wash buffer droplet,a dye/terminator cleavage mix droplet, and a second wash buffer droplet;imaging the plurality of clusters of polynucleotides to detect additionor hybridization of a nucleotide to clusters of the plurality ofclusters of polynucleotides; and repeating the steps of sequentiallymoving each droplet and imaging the plurality of clusters to generatesequence data for the plurality of clusters of polynucleotides.
 14. Themethod of claim 13, further comprising introducing the series of fluidicdroplets into the air gap.
 15. The method of claim 13, whereinsequentially moving each droplet comprises moving each droplet as it isintroduced into the air gap.
 16. The method of claim 13, wherein imagingcomprises imaging the plurality of clusters of polynucleotides aftermoving the first wash buffer droplet over the plurality of clusters ofpolynucleotides.
 17. The method of claim 13, wherein the plurality ofclusters of polynucleotides are arranged in a patterned arrangement. 18.The method of claim 13, wherein moving the one or more mechanical forceapplicators against the first sheet to elastically deform the firstsheet comprises moving one or more styluses against the first sheet. 19.The method of claim 13, further comprising repeatedly applying andreleasing a compression force to the first sheet over the plurality ofclusters of polynucleotides to mix one droplet of the series of fluidicdroplets over the plurality of clusters of polynucleotides.
 20. Themethod of claim 13, wherein sequentially moving each droplet of theseries of fluidic droplets further comprises removing each droplet ofthe series of fluidic droplets from the air gap after it been moved overthe plurality of clusters of polynucleotides.
 21. The method of claim13, wherein each droplet of the series of fluidic droplets has a volumebetween 10⁻⁶ and 10⁻¹⁵ liters.
 22. The method of claim 13, wherein theair gap is open to atmospheric pressure and unpressurized.
 23. Themethod of claim 13, further comprising hybridizing a library ofpolynucleotides to either the first sheet or the second sheet in the airgap to form the clusters of polynucleotides.
 24. A method of sequencingby synthesis using mechanical compression, the method comprising:sequentially introducing a series of fluidic droplets into an air gapformed between: a first sheet having a first surface that is hydrophobicand oleophobic; and a second sheet having a second surface that ishydrophobic and oleophobic, wherein the first sheet and the second sheetare secured opposite at a predetermined spacing relative to each otherwith the air gap therebetween, further comprising a plurality ofclusters of polynucleotides on the second sheet; sequentially movingeach droplet of the series of fluidic droplets over the plurality ofclusters of polynucleotides by moving one or more mechanical forceapplicators against the first sheet to elastically deform the firstsheet and create a region of reduced spacing between the first andsecond sheets so that each droplet follows the region of reduced spacingover the plurality of clusters of polynucleotides, wherein the series offluidic droplets includes: a nucleotide polymerase reaction mix droplet,a first wash buffer droplet, a dye/terminator cleavage mix droplet, anda second wash buffer droplet; imaging the plurality of clusters ofpolynucleotides to detect addition or hybridization of a nucleotide toclusters of the plurality of clusters of polynucleotides; and repeatingthe steps of sequentially introducing the series of fluidic droplets,moving each droplet and imaging the plurality of clusters.